- Email: [email protected]
Seasonal variations of fecal progesterone and 17β-estradiol in captive female black-handed spider monkeys (Ateles geoffroyi)
Theriogenology 66 (2006) 1985–1993 www.journals.elsevierhealth.com/periodicals/the
Seasonal variations of fecal progesterone and 17b-estradiol in captive female black-handed spider monkeys (Ateles geoffroyi) A.L. Cerda-Molina a,*, L. Herna´ndez-Lo´pez a, D.L. Pa´ez-Ponce a, S. Rojas-Maya b, R. Mondrago´n-Ceballos a a
Departamento de Etologı´a, Instituto Nacional de Psiquiatrı´a Ramo´n de la Fuente Mun˜iz, Calzada Me´xico-Xochimilco 101, Col. San Lorenzo Huipulco, Tla´lpan 14370, Me´xico, D.F., Mexico b Departamento de Reproduccio´n de la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Auto´noma de Me´xico, Mexico Received 15 February 2006; accepted 25 March 2006
Abstract A number of studies in free-ranging and captive spider monkeys (Ateles spp.) have shown that this genus is able to reproduce throughout the entire year. Nonetheless, it is still controversial whether births, and therefore conceptions, tend to be more frequent during certain seasons. In the present study, we monitored changes in fecal 17b-estradiol and progesterone for approximately 1 years in five female black-handed spider monkeys (Ateles geoffroyi) kept in captivity in Mexico City. The objective was to determine whether hormone concentrations and menstrual cycles of summer and autumn accounted for a greater chance of conception than those of winter and spring, consistent with birth patterns previously reported. We collected fecal samples from the five monkeys almost daily for 1 year (March 2004 to February 2005) and used radioimmunoassay of fecal extracts to determine concentrations of 17b-estradiol and progesterone. Concurrently, menstrual cycle phases were determined by cytological evaluation of vaginal swabs. Periovulatory 17b-estradiol concentrations were significantly higher in autumn than in winter, spring or summer. Moreover, as evidenced by progesterone peaks, most of the summer–autumn menstrual cycles were ovulatory; in contrast, most of the winter and spring cycles were anovulatory. In conclusion, our data supported the notion that, although not a strictly seasonal reproducer, the black-handed spider monkey is more likely to conceive at the end of the rainy season and throughout autumn. # 2006 Elsevier Inc. All rights reserved. Keywords: Seasonality; Spider monkey; Menstrual cycle; Fecal progesterone; Fecal 17b-estradiol
1. Introduction Most mammalian species exhibit a variety of reproductive strategies involving the integration of
* Corresponding author. Tel.: +52 5 6552811x193 fax: +52 5 6559980. E-mail address: [email protected] (A.L. Cerda-Molina). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.03.038
internal and external stimuli that mediate the sequence of physiological and behavioral events necessary to ensure fertilization. In some species, these events occur at a specific time of the year, so that births occur when food and water are abundant and readily available to lactating females [1]. The birth season varies among species, as do the specific environmental stimuli responsible for the onset of mating activity [2]. Some of the environmental signals commonly involved in
1986
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
seasonal reproductive regulation in mammals are food availability [3], temperature and humidity [2], pheromones [4,5], and day-length (photoperiod) [6]. In primate species, food availability as ultimate cause and photoperiod as proximate cause are the environmental cues assumed to be the most important seasonal regulators of reproduction [3,7,8]. Primate species vary in their degree of seasonality, as does their latitudinal distribution and consequently the quality and availability of food during the year [7,9]. However, near the equator where photoperiod and annual food variations are minimal, mechanisms that time reproductive events are not fully understood [10]. Among the neotropical primates whose pattern of seasonality still remains controversial is the spider monkey genus. For instance, Milton [11] observed two birth-peaks, in autumn and spring, in the black-handed spider monkey (Ateles geoffroyi) population of Barro Colorado, Panama. Fedigan and Rose [12] reported two birth-peaks, winter and spring, in A. geoffroyi kept in Santa Rosa National Park, Costa Rica. In contrast, Klein [13] did not find evidence of birth seasonality in captive A. geoffroyi in La Macarena, Colombia. Further analyses by Chapman and Chapman [14], including many species of captive spider monkeys, supported Klein’s observation of no apparent seasonality, although births tended to occur more frequently in summer and winter. Finally, Di Bitteti and Janson [7] concluded that seasonality in the Ateles genus remains unclear, though their data showed a slight tendency for more births during the dry season. There are no data for seasonal hormone fluctuations in the black-handed spider monkey. The present study is part of an ongoing research on the reproductive biology of this New World monkey. We found that menstrual cycles become irregular at the beginning of spring and that this condition remains until start of autumn [15]. Furthermore, semen samples obtained by electroejaculation in the dry season were of better quality than those obtained in the rainy season [16]. This pattern of semen quality and the regularity of the menstrual cycles during the dry season suggested the onset of ovulatory menstrual cycles during the fall, as well as suppression of cycles in the spring. Consequently, we speculated that black-handed spider monkeys, although not strictly seasonal, are more likely to give birth at specific times of the year and to have seasonal variations in hormone concentrations. Therefore, we conducted a 1 year study of near-daily assessment of fecal 17b-estradiol and progesterone concentrations in females not subjected to stressful manipulations or feeding restrictions. Our hypothesis
was that hormone concentrations would be on average lower in the wet season (spring–summer, when parturition is more likely), but consistent with ovulatory cycles during the fall (dry season). 2. Materials and methods 2.1. Subjects We studied five adult female black-handed spider monkeys, three of them (A–C) approximately 7–9-year old and the other two (D and E) approximately 22–25year old. These animals were housed in a large outdoor enclosure (6.2 m long 1.7 m wide 6 m high), along with three adult males and another three adult females that were too old and uncooperative to be used. Our facility is located at the Ethology Department at the National Institute of Psychiatry in Mexico City. The enclosure is washed daily around 0800 h. Afterwards, animals are fed fresh fruits and vegetables as available according to the season of the year. A supplementary meal of primate chow (Monkey Diet 5038, Lab Diet, PMI Foods, Inc., St. Louis, MO, USA) was provided at mid-day and clean tap water was available ad libitum. 2.2. Fecal sampling Feces were collected at around 1000, 5 or 6 days every week (Monday–Friday and Saturday), for approximately 1 year (March 2004–February 2005). Each experimental animal was fed a bottle (120 mL) of sweetened colored-water (Chefmaster Liqua-gel color, MFG, Byrnes Kiefer Co., Garden Grove, CA, USA) with the mid-day meal; the colors used were red, black, green, yellow, and white. Consequently, we were able to accurately identify the source of the feces. After collection, samples were put into a closed plastic container, labeled, and frozen ( 4 8C) for approximately 3 month until assayed. We collected a total of 800 samples (mean of 41.2 3.0 samples/female/ season). One of the females (C) frequently refused to drink the juice; we lacked data for this female during April and February. 2.3. Hormone extraction and quantification We adapted the technique by Matsumuro et al. [17] for fecal hormone extraction. Feces (2 g) were taken from each plastic container, homogenized for 20 s in 10 mL of distilled water, and left for 18 h at 4 8C. The next morning, samples were centrifuged at 1500 g for 30 min at 4 8C; 5 mL of the supernatant was mixed in
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
10 mL of a 3:2 hexane–ether solution, shaken for 20 s using a vortex mixer, and left on ice for 1 h. Afterwards, 20 mL and 3 mL aliquots were taken for 17b-estradiol and progesterone, respectively. The two aliquots of organic solvents were evaporated to dryness and resuspended in 1.5 mL of phosphate-buffered saline solution (pH 7.5, 50 mM). Estradiol and progesterone were measured with commercially supplied radioimmunoassay kits (125I RIA Kit Coat-A-Count, DPC, Los Angeles, CA, USA). Cross-reactivity provided by the manufacture for the primary antibody was 100% for progesterone, and highly specific for estradiol, with <1% cross-reactivity to other estrogens including estrone, estriol, and their conjugates. Mean recovery rate was determined by adding labeled steroids to fecal samples after they had been homogenized with distilled water—17b-estradiol: 11452 cpm (2, 4, 6, 7-3H Oestradiol, Amersham), specific activity: 3.03 TBq/nmol; progesterone: 9798 cpm (1, 2, 6, 7-3H Progesterone, Amersham), specific activity: 3.40 TBq/nmol. Radioactivity (mCi) was measured using a TRI carb packard counter device (65% efficiency) and a liquid scintillation counter (BETAMAX, ICN Pharmaceuticals, Costa Mesa, CA, USA). The mean percentage (S.E.M.) of 17bestradiol recovered was 30.3 1.8 (n = 18), and 17.1 1.6 (n = 18) for progesterone. The coefficients of intra-assay variation for 17b-estradiol and progesterone were 5.9 and 9.3%, respectively. 2.4. Assessment of menstrual cycle phase In addition to fecal sampling, daily vaginal swabs were taken from all females to assess the menstrual cycle phase. Females were previously trained to allow the introduction of a cotton swab into the vagina to collect vaginal smears. For their cooperation, females were rewarded with cookies. The procedure and interpretation of these data are given in Ref. [15]. Briefly, the cycle phases were classified depending on the proportion of four types of epithelial cells: squamous, superficial, intermediate and parabasal, in addition to the presence of cervical mucus, erythrocytes and lymphocytes [15]. We could accurately determine the cycle’s phases according to the hormone concentrations and day-to-day cytological analyses. Superficial cells increased as the follicular phase progressed and became abundant during the periovulatory period. A predominance of squamous cells during the mid-cycle was the best indicator of the periovulatory phase. We considered the female to be in a luteal phase when the squamous cells disappeared and intermediate cells
1987
proliferated. Finally, menses was confirmed by the abundance of erythrocytes and blood staining of the cotton swab. 2.5. Statistical analyses Mixed linear models for repeated measures were used to analyze seasonal variations of 17b-estradiol and progesterone. The fixed factors were menstrual cycle phases and season, random factors were repeated measurements within female, within menstrual phase, and within season. A first order auto-regressive scheme was used to evaluate random factor covariance. Between-seasons post hoc contrasts were performed by the Bonferroni procedure. The P-level considered as statistically significant for all analyses was 0.05; all tests were two-tailed and we used SPSS 13 to perform the statistical analyses. Seasons were defined as follows: summer, 21 June–22 September; autumn: 23 September–21 December; winter: 22 December–20 March; spring: 21 March–20 June. Additionally, to distinguish anovulatory from ovulatory cycles, we followed Ziegler et al. [18]; they considered luteal progesterone peaks 3S.D. higher than the overall mean (by female) as ovulatory. 3. Results Fecal hormone variations throughout the 1-year period for four spider monkey females (A–D) are illustrated in Figs. 1 and 2. There was an apparent tendency for 17b-estradiol to reach peaks >100 pg/g in the last days of September, approximately corresponding to the onset of the dry season (Fig. 1). These peaks continued from October to December, except for Female B (cycling pattern continued to February). Estradiol concentrations were lowest (on average 50 pg/g) from March to August (i.e. spring and summer) in Females A–C. In contrast, in Female D, estradiol concentrations were lowest from November to March, and she had intermediate (>50 and <100 pg/g) and various high peaks of estradiol (>100 pg/g) throughout spring and summer (Fig. 1D). Compared to estradiol, overall and peak concentrations of progesterone were more variable among females. Female A had relatively low progesterone concentrations throughout spring and early summer, then, in the first days of July she had a large progesterone peak (19.02 ng/g), with similar peaks until late September, past the onset of fall (Fig. 2A). Five peaks were identified as belonging to ovulatory cycles (Table 1). Female B (Fig. 2B) had progesterone
1988
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Fig. 1. Fecal concentrations of 17b-estradiol in three female spider monkeys from March 2004 to February 2005. The rainy season started in June and continued to mid-September.
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
1989
Fig. 2. Fecal concentrations of progesterone in three female spider monkeys from March 2004 to February 2005. The rainy season started in June and continued to mid-September.
1990
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Table 1 Occurrence of fecal progesterone peaks >3S.D. above the mean (of each female) in captive female black-handed spider monkeys Female
Month
Date
Progesterone (ng/g)
Season
A A A A A C C C C E E E E B B B D A A C C E E B B B B B D D D D B D D
July July August September September August August August September July August September September August September September September September October September October October October March April April May May May May May May March February March
06 July 2004 29 July 2004 20 August 2004 14 September 2004 15 September 2004 10 August 2004 11 August 2004 20 August 2004 07 September 2004 13 July 2004 10 August 2004 01 September 2004 02 September 2004 17 August 2004 10 September 2004 21 September 2004 14 September 2004 28 September 2004 01 October 2004 24 September 2004 07 October 2004 01 October 2004 05 October 2004 11 March 2004 28 April 2004 29 April 2004 07 May 2004 21 May 2004 17 May 2004 19 May 2004 20 May 2004 21 May 2004 19 March 2005 19 February 2004 9 March 2004
19.0 9.7 14.6 13.8 12.3 7.4 7.8 12.4 8.4 34.1 40.0 40.0 40.0 31.1 27.2 22.5 20.5 17.3 11.7 8.5 8.2 40.0 40.0 19.6 18.9 23.6 20.2 21.2 23.9 40.0 21.7 21.0 20.5 40.0 40.0
Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Autumn Autumn Autumn Autumn Autumn Autumn Spring Spring Spring Spring Spring Spring Spring Spring Spring Winter Winter Winter
Most progesterone peaks occurred from May to September (rainy season). Two females, the youngest one (B) and the oldest one (D), also had progesterone peaks from February to April.
peaks from March to May (spring), from August to October (mid-summer to fall), and finally, at the end of the sampling period, mid-March 2005 (almost spring), she again seemed to have increasing progesterone concentrations. Five ovulatory peaks were identified in the first peaking period (spring), whereas only three (just those during the summer), belonged to ovulatory cycles in the second peaking period (Table 1). Finally, the last progesterone peak for female B (at the end of the sampling period) also belonged to an ovulatory cycle. Female C had lower overall progesterone concentrations than the remaining three females. Nonetheless, she had progesterone peaks from July to November (Fig. 2C). Of these (Table 1), those occurring during summer and early autumn belonged to ovulatory cycles.
Female D had progesterone peaks throughout all of 2004 until October (Fig. 2D). Thereafter, she had low concentrations for 3 month, followed by an abrupt increase in February 2005. Five peaks from March to May occurred within ovulatory cycles (Table 1), but only a single peak (mid-September) was ovulatory. Female E is shown independently (Fig. 3), as she was artificially inseminated on 26 October; thereafter, her endocrine concentrations and patterns changed drastically. Data for this female are presented on a logarithmic scale. There were two periods of estradiol and progesterone peaks; a first estradiol peak period started in September, continued until October (>100 pg/g) and ended the day of artificial insemination (458 pg/g). From March to August, estradiol
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
1991
Fig. 3. Fecal concentrations (logarithmic scale) of 17b-estradiol and progesterone of one female spider monkey (E) from March 2004 to February 2005. The arrow indicated the day of artificial insemination.
concentrations were <100 pg/g. Progesterone first peaked at the end of March and continued until May (20 ng/g). On the other hand, progesterone increased and peaked before estradiol in the second period; it started in mid-July, almost 2 month earlier (< 40 ng/ g). Progesterone peaks ended with artificial insemination, abruptly increased by late November due to pregnancy, and reached concentrations as high as 2000 ng/g. Nonetheless, only the summertime peaks were of an ovulatory nature (Table 1). There was no effect of age on estradiol concentrations; although Females D and E were the oldest animals, the maximum peak in female E was the highest value (458 pg/g). Conversely, progesterone concentrations throughout the year were similar among females (5 ng/g), but progesterone peaks were higher in the older females (reached 40 ng/g).
Regarding seasonal variations for all five females, fecal 17b-estradiol concentrations significantly changed across menstrual phases (F 3,192.3 = 50.313, P < 0.001) and seasons (F 3,62.9 = 6.567, P < 0.001); the season menstrual cycle phase was also significant (F9, 64.2 = 4.037, P < 0.001). Fig. 4 shows the season menstrual cycle phase interaction mean effects. Post hoc contrasts revealed that mean (S.E.M.) 17bestradiol concentrations in autumn (113.4 7 pg/g) were significantly higher (P < 0.05) than those in spring (55.7 8 pg/g), summer (55.7 6 pg/g), and winter (65.3 10.1 pg/g) but only for the periovulatory phase of the cycle (Fig. 4). Progesterone did not differ across seasons (F 3,71.40 = 1.18, P = NS) nor did the season x menstrual cycle interaction (F9, 68.44 = 1.48, P = NS), and only fluctuations within menstrual cycle phases had significant changes (F 3,198.11 = 4.12, P < 0.001).
Fig. 4. Mean (S.E.M.) fecal 17b-estradiol concentrations collected during 1 year from five female spider monkeys. Periovulatory concentrations of 17b-estradiol were higher during the autumn than in any other season (*P < 0.05).
1992
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
This was mainly due to the immense within-day-to-day variation of progesterone concentrations, ranging from 0.01 to >40 ng/g in all females. Nonetheless, progesterone peaks (3S.D.) occurred mostly in the summer for all five females, although two females (B and D) also had some peaks in the autumn and winter (Table 1). 4. Discussion The present study provided further evidence that captive female black-handed spider monkeys, although not strictly seasonal, have hormone variations minimizing or maximizing reproductive success during specific seasons of the year. Measurement of fecal 17b-estradiol and progesterone are well accepted as a non-invasive means of determining the reproductive status of female primates [19]. Other authors reported exceedingly high concentrations of hormones in the black-handed spider monkeys [20,21]; although our recovery rate was very low, hormone fluctuations were readily apparent in all females. Progesterone concentrations were higher in old versus young females. There are no indications of reproductive senescence in female spider monkeys; furthermore, Milton [11], reported that black-handed spider monkey females >20-year old could be fertile. We suggest that instead of physiological process related to age, hormonal differences may be related to social rank [22], as Females D and E were the dominant females of the group. In addition, even though only five females were used, seasonal variations in fecal 17b-estradiol concentrations were consistent among animals. Low 17bestradiol concentrations and small progesterone peaks were characteristic of winter and spring, consistent with anovulatory menstrual cycles. Conversely 17b-estradiol concentrations were highest during the autumn, whereas the uppermost progesterone peaks started appearing in late summer and throughout the autumn. Goodman [1] stated that for seasonally reproducing mammals that give birth throughout spring and summer, food availability is the ultimate factor accounting for seasonality, an idea further advanced by Di Bitetti and Janson [7] for Neotropical primates. This pattern would allow dams to sustain the first 3 month of lactation; thereafter, their offspring would supplement their diet by eating non-milk food items. According to our studies, autumn would be the ideal time for conception, as most ovulatory cycles take place in this season (high estrogen concentrations in females) and semen quality in males are maximal [16]. That the duration of gestation in the black-handed
spider monkey is approximately 226–232 days [23] or exactly 210 days (Herna´ndez-Lo´pez et al., submitted for publication), birthing would commence with the start of spring (end of March and beginning of April), and continue until mid-summer (July–August). This conclusion was consistent with Di Bitetti and Janson [7] and others [24,1], suggesting that the most common seasonal pattern found in New World primates is mating and conception at the beginning of the dry season, with parturition coinciding with abundant food. Regarding the availability of food (in particular fruit) shortages should not be common, as the fruit species consumed by Mexican spider monkeys either produce fruit all year long (Ficus spp.), or alternately throughout the year [25]. Nevertheless, the palatability and nutritional quality of fruits do change with seasons; there is a greater diversity of mature fruits available in the spring (March to early June) [25], and female spider monkeys are particularly behavior-sensitive to changes in their diet [23]. In Mexico City (latitude, 198N and elevation, 2235 m), autumn marks the end of the rainy season and the beginning of the dry season; the rainy season usually starts at the end of spring. In the eastern (Yucata´n Peninsula), south east (Veracruz and Tabasco) and southern parts of Mexico (Chiapas and Oaxaca), where wild populations of spider monkeys are found, dry and rainy seasons occur at corresponding times, although temperature variations are less evident [26,27]. Along with the onset of the rainy and dry seasons, changes in day-length occur in the Tropic of Cancer, where the Mexican monkey populations are located. The duration of daylight increases on average 1 h during the summer (and decreases 1 h during the winter). Although the current study was performed in captive animals, the present results, along with seasonal variations in sperm quality in males [16], were consistent with the above-mentioned environmental variations, and seemed to represent true adaptation rather than artifacts of confinement. It was noteworthy that seasonality appeared in captivity (where the food supply was not limited). Furthermore, it was reported [14] that for animals dwelling in northern latitudes, daylength, rather than humidity or vegetation cues, apparently synchronized reproductive events. In summary, fecal hormone concentrations of female spider monkeys tend to vary seasonally, with lower concentrations from the end of the dry season to the wet season (i.e. spring–summer), with the highest concentrations during the beginning of the dry season (i.e. fall).
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Acknowledgements This research project was supported by the Instituto Nacional de Psiquiatrı´a ‘‘Ramo´n de la Fuente Mun˜iz’’. We are grateful to Roberto Chavira for performing the recovery hormone test. We thank Isabel Pe´rez Montfort for correcting the English version of the manuscript. We are grateful to two anonymous reviewers who provided comments that greatly improved this paper. References [1] Goodman R. Seasonal Reproduction, Mammals. In: Knobil E, Neill JD, editors. Encyclopedia of reproduction. New York: Academic Press; 1999. p. 341–51. [2] Bronson FH. Seasonal regulation of reproduction in mammals. In: Knobil E, Neill J, editors. The physiology of reproduction. New York: Raven Press Ltd.; 1988. p. 1831–73. [3] Koening A, Borrie C, Chalise MK, Winkler P. Ecology, nutrition, and the timing of reproductive events in an Asian primate, the Hanuman langur (Presbytis entellus). J Zool (London) 1997; 243:215–35. [4] Bronson FH, Macmillan B. Hormonal responses to primer pheromones. In: Vandenbergh JG, editor. Pheromones and reproduction in mammals. New York: Academic Press; 1983. p. 175–97. [5] Signoret JP. Sexual pheromones in the domestic sheep: importance and limits in the regulation of reproductive physiology. J Steroid Biochem Mol Biol 1991;39:639–45. [6] Hastings MH, Herbert J, Martensz ND, Roberts AC. Annual reproductive rhythms in mammals: mechanism of light synchronization. Ann NY Acad Sci 1985;453:182–204. [7] Di Bitetti MS, Janson CH. When will the stork arrive? Patterns of birth seasonality in neotropical primates. Am J Primatol 2000; 50:109–30. [8] Anderson DP, Nordheim EV, Boesch C. Environmental factors influencing the seasonality of estrus in chimpanzees. Primates 2006;47:43–50. [9] Bicca-Marques JC, Fichtner D. Birth seasonality of Cebus paella (Platyrrhini, Cebidae) in Brazilian Zoos along a latitudinal gradient. Am J Primatol 2005;65:141–7. [10] Strier KB, Mendes SL, Santos RR. Timing of births in sympatric brown howler monkeys (Alouatta fusca clamitans) and northern muriquis (Brachyteles arachnoides hypoxanthus). Am J Primatol 2001;55:87–100. [11] Milton K. Estimates of reproductive parameters for free-ranging Ateles geoffroyi. Primates 1981;22:574–9. [12] Fedigan LM, Rose LM. Interbirth interval variation in three sympatric species of neotropical monkey. Am J Primatol 1995; 37:9–24.
1993
[13] Klein LL. Observations on copulations and seasonal reproduction of two species of spider monkeys, Ateles belzebuth and A. geoffroyi. Fol Primatol 1971;15:233–48. [14] Chapman C, Chapman L. Reproductive biology of captive and free-ranging spider monkeys. Zoo Biol 1990;9:1–9. [15] Herna´ndez-Lo´pez L, Mayagoitia L, Esquivel-Lacroix C, RojasMaya S, Mondrago´n-Ceballos R. The menstrual cycle of the spider monkey (Ateles geoffroyi). Am J Primatol 1998;44:183– 95. [16] Herna´ndez-Lo´pez L, Cerezo-Parra G, Cerda-Molina AL, Pe´rezBolan˜os SC, Dı´az-Sa´nchez V, Mondrago´n-Ceballos R. Sperm quality differences between the rainy and dry seasons in captive black-handed spider monkeys (Ateles geoffroyi). Am J Primatol 2002;57:35–41. [17] Matsumuro M, Tadashi S, Fumiaki C, Yasuhiro Y, Takashi Y. A two-step extraction method to measure fecal hormones in female cynomulgus monkey (Macaca fascicularis). Am J Primatol 1999;48:291–8. [18] Ziegler T, Hodges K, Winkler P, Heistermann M. Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus). Am J Primatol 2000;51: 119–34. [19] Strier KB, Ziegler TE. Behavioral and endocrine characteristics of the reproductive cycle in wild muriqui monkeys, Brachyteles arachnoids. Am J Primatol 1997;42:299–310. [20] Campbell C, Shideler SE, Todd HE, Lasley BL. Fecal analysis of ovarian cycles in female black-handed spider monkeys (Ateles geoffroyi). Am J Primatol 2001;54:79–89. [21] Campbell C. Patterns of behavior across reproductive states of free-ranging female black-handed spider monkeys (Ateles geoffroyi). Am J Phys Anthropol 2004;124:166–76. [22] Dixson AF, Primate Sexuality. Comparative studies of the prosimians, monkeys, apes and human beings Oxford: Oxford University Press; 1998. p. 444–53. [23] Eisenberg JF. Communication mechanism and social integration in the black spider monkey, Ateles fusciceps robustus, and related species. Smithson Contrib Zool 1976;213:1–108. [24] Van Roosmalen MG, M Klein L. The spider monkeys, genus Ateles. In: Mittermeier R, Rylands A, editors. Ecology and behavior of neotropical primates. Washington, DC: World Wildlife Fund; 1988. p. 455–537. [25] Pe´rez-Ruiz AL. Disponibilidad de alimento y comportamiento afiliativo del mono aran˜a (Ateles geoffroyi) en la selva lacandona, Chiapas. Master Degree Thesis; 2004. [26] Estrada A, Coates-Estrada R. Some observations on the present distribution and conservation of Alouatta and Ateles in southern Mexico. Am J Primatol 1984;7:133–7. [27] Goldizen AW, Tergorgh J, Cornejo F, Porras DT, Evans R. Seasonal food shortage, weight loss, and the timing of births in saddle-back tamarins (Saguinus fuscicollis). J Anim Ecol 1988;57:893–901.
Seasonal variations of fecal progesterone and 17b-estradiol in captive female black-handed spider monkeys (Ateles geoffroyi) A.L. Cerda-Molina a,*, L. Herna´ndez-Lo´pez a, D.L. Pa´ez-Ponce a, S. Rojas-Maya b, R. Mondrago´n-Ceballos a a
Departamento de Etologı´a, Instituto Nacional de Psiquiatrı´a Ramo´n de la Fuente Mun˜iz, Calzada Me´xico-Xochimilco 101, Col. San Lorenzo Huipulco, Tla´lpan 14370, Me´xico, D.F., Mexico b Departamento de Reproduccio´n de la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Nacional Auto´noma de Me´xico, Mexico Received 15 February 2006; accepted 25 March 2006
Abstract A number of studies in free-ranging and captive spider monkeys (Ateles spp.) have shown that this genus is able to reproduce throughout the entire year. Nonetheless, it is still controversial whether births, and therefore conceptions, tend to be more frequent during certain seasons. In the present study, we monitored changes in fecal 17b-estradiol and progesterone for approximately 1 years in five female black-handed spider monkeys (Ateles geoffroyi) kept in captivity in Mexico City. The objective was to determine whether hormone concentrations and menstrual cycles of summer and autumn accounted for a greater chance of conception than those of winter and spring, consistent with birth patterns previously reported. We collected fecal samples from the five monkeys almost daily for 1 year (March 2004 to February 2005) and used radioimmunoassay of fecal extracts to determine concentrations of 17b-estradiol and progesterone. Concurrently, menstrual cycle phases were determined by cytological evaluation of vaginal swabs. Periovulatory 17b-estradiol concentrations were significantly higher in autumn than in winter, spring or summer. Moreover, as evidenced by progesterone peaks, most of the summer–autumn menstrual cycles were ovulatory; in contrast, most of the winter and spring cycles were anovulatory. In conclusion, our data supported the notion that, although not a strictly seasonal reproducer, the black-handed spider monkey is more likely to conceive at the end of the rainy season and throughout autumn. # 2006 Elsevier Inc. All rights reserved. Keywords: Seasonality; Spider monkey; Menstrual cycle; Fecal progesterone; Fecal 17b-estradiol
1. Introduction Most mammalian species exhibit a variety of reproductive strategies involving the integration of
* Corresponding author. Tel.: +52 5 6552811x193 fax: +52 5 6559980. E-mail address: [email protected] (A.L. Cerda-Molina). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.03.038
internal and external stimuli that mediate the sequence of physiological and behavioral events necessary to ensure fertilization. In some species, these events occur at a specific time of the year, so that births occur when food and water are abundant and readily available to lactating females [1]. The birth season varies among species, as do the specific environmental stimuli responsible for the onset of mating activity [2]. Some of the environmental signals commonly involved in
1986
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
seasonal reproductive regulation in mammals are food availability [3], temperature and humidity [2], pheromones [4,5], and day-length (photoperiod) [6]. In primate species, food availability as ultimate cause and photoperiod as proximate cause are the environmental cues assumed to be the most important seasonal regulators of reproduction [3,7,8]. Primate species vary in their degree of seasonality, as does their latitudinal distribution and consequently the quality and availability of food during the year [7,9]. However, near the equator where photoperiod and annual food variations are minimal, mechanisms that time reproductive events are not fully understood [10]. Among the neotropical primates whose pattern of seasonality still remains controversial is the spider monkey genus. For instance, Milton [11] observed two birth-peaks, in autumn and spring, in the black-handed spider monkey (Ateles geoffroyi) population of Barro Colorado, Panama. Fedigan and Rose [12] reported two birth-peaks, winter and spring, in A. geoffroyi kept in Santa Rosa National Park, Costa Rica. In contrast, Klein [13] did not find evidence of birth seasonality in captive A. geoffroyi in La Macarena, Colombia. Further analyses by Chapman and Chapman [14], including many species of captive spider monkeys, supported Klein’s observation of no apparent seasonality, although births tended to occur more frequently in summer and winter. Finally, Di Bitteti and Janson [7] concluded that seasonality in the Ateles genus remains unclear, though their data showed a slight tendency for more births during the dry season. There are no data for seasonal hormone fluctuations in the black-handed spider monkey. The present study is part of an ongoing research on the reproductive biology of this New World monkey. We found that menstrual cycles become irregular at the beginning of spring and that this condition remains until start of autumn [15]. Furthermore, semen samples obtained by electroejaculation in the dry season were of better quality than those obtained in the rainy season [16]. This pattern of semen quality and the regularity of the menstrual cycles during the dry season suggested the onset of ovulatory menstrual cycles during the fall, as well as suppression of cycles in the spring. Consequently, we speculated that black-handed spider monkeys, although not strictly seasonal, are more likely to give birth at specific times of the year and to have seasonal variations in hormone concentrations. Therefore, we conducted a 1 year study of near-daily assessment of fecal 17b-estradiol and progesterone concentrations in females not subjected to stressful manipulations or feeding restrictions. Our hypothesis
was that hormone concentrations would be on average lower in the wet season (spring–summer, when parturition is more likely), but consistent with ovulatory cycles during the fall (dry season). 2. Materials and methods 2.1. Subjects We studied five adult female black-handed spider monkeys, three of them (A–C) approximately 7–9-year old and the other two (D and E) approximately 22–25year old. These animals were housed in a large outdoor enclosure (6.2 m long 1.7 m wide 6 m high), along with three adult males and another three adult females that were too old and uncooperative to be used. Our facility is located at the Ethology Department at the National Institute of Psychiatry in Mexico City. The enclosure is washed daily around 0800 h. Afterwards, animals are fed fresh fruits and vegetables as available according to the season of the year. A supplementary meal of primate chow (Monkey Diet 5038, Lab Diet, PMI Foods, Inc., St. Louis, MO, USA) was provided at mid-day and clean tap water was available ad libitum. 2.2. Fecal sampling Feces were collected at around 1000, 5 or 6 days every week (Monday–Friday and Saturday), for approximately 1 year (March 2004–February 2005). Each experimental animal was fed a bottle (120 mL) of sweetened colored-water (Chefmaster Liqua-gel color, MFG, Byrnes Kiefer Co., Garden Grove, CA, USA) with the mid-day meal; the colors used were red, black, green, yellow, and white. Consequently, we were able to accurately identify the source of the feces. After collection, samples were put into a closed plastic container, labeled, and frozen ( 4 8C) for approximately 3 month until assayed. We collected a total of 800 samples (mean of 41.2 3.0 samples/female/ season). One of the females (C) frequently refused to drink the juice; we lacked data for this female during April and February. 2.3. Hormone extraction and quantification We adapted the technique by Matsumuro et al. [17] for fecal hormone extraction. Feces (2 g) were taken from each plastic container, homogenized for 20 s in 10 mL of distilled water, and left for 18 h at 4 8C. The next morning, samples were centrifuged at 1500 g for 30 min at 4 8C; 5 mL of the supernatant was mixed in
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
10 mL of a 3:2 hexane–ether solution, shaken for 20 s using a vortex mixer, and left on ice for 1 h. Afterwards, 20 mL and 3 mL aliquots were taken for 17b-estradiol and progesterone, respectively. The two aliquots of organic solvents were evaporated to dryness and resuspended in 1.5 mL of phosphate-buffered saline solution (pH 7.5, 50 mM). Estradiol and progesterone were measured with commercially supplied radioimmunoassay kits (125I RIA Kit Coat-A-Count, DPC, Los Angeles, CA, USA). Cross-reactivity provided by the manufacture for the primary antibody was 100% for progesterone, and highly specific for estradiol, with <1% cross-reactivity to other estrogens including estrone, estriol, and their conjugates. Mean recovery rate was determined by adding labeled steroids to fecal samples after they had been homogenized with distilled water—17b-estradiol: 11452 cpm (2, 4, 6, 7-3H Oestradiol, Amersham), specific activity: 3.03 TBq/nmol; progesterone: 9798 cpm (1, 2, 6, 7-3H Progesterone, Amersham), specific activity: 3.40 TBq/nmol. Radioactivity (mCi) was measured using a TRI carb packard counter device (65% efficiency) and a liquid scintillation counter (BETAMAX, ICN Pharmaceuticals, Costa Mesa, CA, USA). The mean percentage (S.E.M.) of 17bestradiol recovered was 30.3 1.8 (n = 18), and 17.1 1.6 (n = 18) for progesterone. The coefficients of intra-assay variation for 17b-estradiol and progesterone were 5.9 and 9.3%, respectively. 2.4. Assessment of menstrual cycle phase In addition to fecal sampling, daily vaginal swabs were taken from all females to assess the menstrual cycle phase. Females were previously trained to allow the introduction of a cotton swab into the vagina to collect vaginal smears. For their cooperation, females were rewarded with cookies. The procedure and interpretation of these data are given in Ref. [15]. Briefly, the cycle phases were classified depending on the proportion of four types of epithelial cells: squamous, superficial, intermediate and parabasal, in addition to the presence of cervical mucus, erythrocytes and lymphocytes [15]. We could accurately determine the cycle’s phases according to the hormone concentrations and day-to-day cytological analyses. Superficial cells increased as the follicular phase progressed and became abundant during the periovulatory period. A predominance of squamous cells during the mid-cycle was the best indicator of the periovulatory phase. We considered the female to be in a luteal phase when the squamous cells disappeared and intermediate cells
1987
proliferated. Finally, menses was confirmed by the abundance of erythrocytes and blood staining of the cotton swab. 2.5. Statistical analyses Mixed linear models for repeated measures were used to analyze seasonal variations of 17b-estradiol and progesterone. The fixed factors were menstrual cycle phases and season, random factors were repeated measurements within female, within menstrual phase, and within season. A first order auto-regressive scheme was used to evaluate random factor covariance. Between-seasons post hoc contrasts were performed by the Bonferroni procedure. The P-level considered as statistically significant for all analyses was 0.05; all tests were two-tailed and we used SPSS 13 to perform the statistical analyses. Seasons were defined as follows: summer, 21 June–22 September; autumn: 23 September–21 December; winter: 22 December–20 March; spring: 21 March–20 June. Additionally, to distinguish anovulatory from ovulatory cycles, we followed Ziegler et al. [18]; they considered luteal progesterone peaks 3S.D. higher than the overall mean (by female) as ovulatory. 3. Results Fecal hormone variations throughout the 1-year period for four spider monkey females (A–D) are illustrated in Figs. 1 and 2. There was an apparent tendency for 17b-estradiol to reach peaks >100 pg/g in the last days of September, approximately corresponding to the onset of the dry season (Fig. 1). These peaks continued from October to December, except for Female B (cycling pattern continued to February). Estradiol concentrations were lowest (on average 50 pg/g) from March to August (i.e. spring and summer) in Females A–C. In contrast, in Female D, estradiol concentrations were lowest from November to March, and she had intermediate (>50 and <100 pg/g) and various high peaks of estradiol (>100 pg/g) throughout spring and summer (Fig. 1D). Compared to estradiol, overall and peak concentrations of progesterone were more variable among females. Female A had relatively low progesterone concentrations throughout spring and early summer, then, in the first days of July she had a large progesterone peak (19.02 ng/g), with similar peaks until late September, past the onset of fall (Fig. 2A). Five peaks were identified as belonging to ovulatory cycles (Table 1). Female B (Fig. 2B) had progesterone
1988
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Fig. 1. Fecal concentrations of 17b-estradiol in three female spider monkeys from March 2004 to February 2005. The rainy season started in June and continued to mid-September.
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
1989
Fig. 2. Fecal concentrations of progesterone in three female spider monkeys from March 2004 to February 2005. The rainy season started in June and continued to mid-September.
1990
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Table 1 Occurrence of fecal progesterone peaks >3S.D. above the mean (of each female) in captive female black-handed spider monkeys Female
Month
Date
Progesterone (ng/g)
Season
A A A A A C C C C E E E E B B B D A A C C E E B B B B B D D D D B D D
July July August September September August August August September July August September September August September September September September October September October October October March April April May May May May May May March February March
06 July 2004 29 July 2004 20 August 2004 14 September 2004 15 September 2004 10 August 2004 11 August 2004 20 August 2004 07 September 2004 13 July 2004 10 August 2004 01 September 2004 02 September 2004 17 August 2004 10 September 2004 21 September 2004 14 September 2004 28 September 2004 01 October 2004 24 September 2004 07 October 2004 01 October 2004 05 October 2004 11 March 2004 28 April 2004 29 April 2004 07 May 2004 21 May 2004 17 May 2004 19 May 2004 20 May 2004 21 May 2004 19 March 2005 19 February 2004 9 March 2004
19.0 9.7 14.6 13.8 12.3 7.4 7.8 12.4 8.4 34.1 40.0 40.0 40.0 31.1 27.2 22.5 20.5 17.3 11.7 8.5 8.2 40.0 40.0 19.6 18.9 23.6 20.2 21.2 23.9 40.0 21.7 21.0 20.5 40.0 40.0
Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Summer Autumn Autumn Autumn Autumn Autumn Autumn Spring Spring Spring Spring Spring Spring Spring Spring Spring Winter Winter Winter
Most progesterone peaks occurred from May to September (rainy season). Two females, the youngest one (B) and the oldest one (D), also had progesterone peaks from February to April.
peaks from March to May (spring), from August to October (mid-summer to fall), and finally, at the end of the sampling period, mid-March 2005 (almost spring), she again seemed to have increasing progesterone concentrations. Five ovulatory peaks were identified in the first peaking period (spring), whereas only three (just those during the summer), belonged to ovulatory cycles in the second peaking period (Table 1). Finally, the last progesterone peak for female B (at the end of the sampling period) also belonged to an ovulatory cycle. Female C had lower overall progesterone concentrations than the remaining three females. Nonetheless, she had progesterone peaks from July to November (Fig. 2C). Of these (Table 1), those occurring during summer and early autumn belonged to ovulatory cycles.
Female D had progesterone peaks throughout all of 2004 until October (Fig. 2D). Thereafter, she had low concentrations for 3 month, followed by an abrupt increase in February 2005. Five peaks from March to May occurred within ovulatory cycles (Table 1), but only a single peak (mid-September) was ovulatory. Female E is shown independently (Fig. 3), as she was artificially inseminated on 26 October; thereafter, her endocrine concentrations and patterns changed drastically. Data for this female are presented on a logarithmic scale. There were two periods of estradiol and progesterone peaks; a first estradiol peak period started in September, continued until October (>100 pg/g) and ended the day of artificial insemination (458 pg/g). From March to August, estradiol
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
1991
Fig. 3. Fecal concentrations (logarithmic scale) of 17b-estradiol and progesterone of one female spider monkey (E) from March 2004 to February 2005. The arrow indicated the day of artificial insemination.
concentrations were <100 pg/g. Progesterone first peaked at the end of March and continued until May (20 ng/g). On the other hand, progesterone increased and peaked before estradiol in the second period; it started in mid-July, almost 2 month earlier (< 40 ng/ g). Progesterone peaks ended with artificial insemination, abruptly increased by late November due to pregnancy, and reached concentrations as high as 2000 ng/g. Nonetheless, only the summertime peaks were of an ovulatory nature (Table 1). There was no effect of age on estradiol concentrations; although Females D and E were the oldest animals, the maximum peak in female E was the highest value (458 pg/g). Conversely, progesterone concentrations throughout the year were similar among females (5 ng/g), but progesterone peaks were higher in the older females (reached 40 ng/g).
Regarding seasonal variations for all five females, fecal 17b-estradiol concentrations significantly changed across menstrual phases (F 3,192.3 = 50.313, P < 0.001) and seasons (F 3,62.9 = 6.567, P < 0.001); the season menstrual cycle phase was also significant (F9, 64.2 = 4.037, P < 0.001). Fig. 4 shows the season menstrual cycle phase interaction mean effects. Post hoc contrasts revealed that mean (S.E.M.) 17bestradiol concentrations in autumn (113.4 7 pg/g) were significantly higher (P < 0.05) than those in spring (55.7 8 pg/g), summer (55.7 6 pg/g), and winter (65.3 10.1 pg/g) but only for the periovulatory phase of the cycle (Fig. 4). Progesterone did not differ across seasons (F 3,71.40 = 1.18, P = NS) nor did the season x menstrual cycle interaction (F9, 68.44 = 1.48, P = NS), and only fluctuations within menstrual cycle phases had significant changes (F 3,198.11 = 4.12, P < 0.001).
Fig. 4. Mean (S.E.M.) fecal 17b-estradiol concentrations collected during 1 year from five female spider monkeys. Periovulatory concentrations of 17b-estradiol were higher during the autumn than in any other season (*P < 0.05).
1992
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
This was mainly due to the immense within-day-to-day variation of progesterone concentrations, ranging from 0.01 to >40 ng/g in all females. Nonetheless, progesterone peaks (3S.D.) occurred mostly in the summer for all five females, although two females (B and D) also had some peaks in the autumn and winter (Table 1). 4. Discussion The present study provided further evidence that captive female black-handed spider monkeys, although not strictly seasonal, have hormone variations minimizing or maximizing reproductive success during specific seasons of the year. Measurement of fecal 17b-estradiol and progesterone are well accepted as a non-invasive means of determining the reproductive status of female primates [19]. Other authors reported exceedingly high concentrations of hormones in the black-handed spider monkeys [20,21]; although our recovery rate was very low, hormone fluctuations were readily apparent in all females. Progesterone concentrations were higher in old versus young females. There are no indications of reproductive senescence in female spider monkeys; furthermore, Milton [11], reported that black-handed spider monkey females >20-year old could be fertile. We suggest that instead of physiological process related to age, hormonal differences may be related to social rank [22], as Females D and E were the dominant females of the group. In addition, even though only five females were used, seasonal variations in fecal 17b-estradiol concentrations were consistent among animals. Low 17bestradiol concentrations and small progesterone peaks were characteristic of winter and spring, consistent with anovulatory menstrual cycles. Conversely 17b-estradiol concentrations were highest during the autumn, whereas the uppermost progesterone peaks started appearing in late summer and throughout the autumn. Goodman [1] stated that for seasonally reproducing mammals that give birth throughout spring and summer, food availability is the ultimate factor accounting for seasonality, an idea further advanced by Di Bitetti and Janson [7] for Neotropical primates. This pattern would allow dams to sustain the first 3 month of lactation; thereafter, their offspring would supplement their diet by eating non-milk food items. According to our studies, autumn would be the ideal time for conception, as most ovulatory cycles take place in this season (high estrogen concentrations in females) and semen quality in males are maximal [16]. That the duration of gestation in the black-handed
spider monkey is approximately 226–232 days [23] or exactly 210 days (Herna´ndez-Lo´pez et al., submitted for publication), birthing would commence with the start of spring (end of March and beginning of April), and continue until mid-summer (July–August). This conclusion was consistent with Di Bitetti and Janson [7] and others [24,1], suggesting that the most common seasonal pattern found in New World primates is mating and conception at the beginning of the dry season, with parturition coinciding with abundant food. Regarding the availability of food (in particular fruit) shortages should not be common, as the fruit species consumed by Mexican spider monkeys either produce fruit all year long (Ficus spp.), or alternately throughout the year [25]. Nevertheless, the palatability and nutritional quality of fruits do change with seasons; there is a greater diversity of mature fruits available in the spring (March to early June) [25], and female spider monkeys are particularly behavior-sensitive to changes in their diet [23]. In Mexico City (latitude, 198N and elevation, 2235 m), autumn marks the end of the rainy season and the beginning of the dry season; the rainy season usually starts at the end of spring. In the eastern (Yucata´n Peninsula), south east (Veracruz and Tabasco) and southern parts of Mexico (Chiapas and Oaxaca), where wild populations of spider monkeys are found, dry and rainy seasons occur at corresponding times, although temperature variations are less evident [26,27]. Along with the onset of the rainy and dry seasons, changes in day-length occur in the Tropic of Cancer, where the Mexican monkey populations are located. The duration of daylight increases on average 1 h during the summer (and decreases 1 h during the winter). Although the current study was performed in captive animals, the present results, along with seasonal variations in sperm quality in males [16], were consistent with the above-mentioned environmental variations, and seemed to represent true adaptation rather than artifacts of confinement. It was noteworthy that seasonality appeared in captivity (where the food supply was not limited). Furthermore, it was reported [14] that for animals dwelling in northern latitudes, daylength, rather than humidity or vegetation cues, apparently synchronized reproductive events. In summary, fecal hormone concentrations of female spider monkeys tend to vary seasonally, with lower concentrations from the end of the dry season to the wet season (i.e. spring–summer), with the highest concentrations during the beginning of the dry season (i.e. fall).
A.L. Cerda-Molina et al. / Theriogenology 66 (2006) 1985–1993
Acknowledgements This research project was supported by the Instituto Nacional de Psiquiatrı´a ‘‘Ramo´n de la Fuente Mun˜iz’’. We are grateful to Roberto Chavira for performing the recovery hormone test. We thank Isabel Pe´rez Montfort for correcting the English version of the manuscript. We are grateful to two anonymous reviewers who provided comments that greatly improved this paper. References [1] Goodman R. Seasonal Reproduction, Mammals. In: Knobil E, Neill JD, editors. Encyclopedia of reproduction. New York: Academic Press; 1999. p. 341–51. [2] Bronson FH. Seasonal regulation of reproduction in mammals. In: Knobil E, Neill J, editors. The physiology of reproduction. New York: Raven Press Ltd.; 1988. p. 1831–73. [3] Koening A, Borrie C, Chalise MK, Winkler P. Ecology, nutrition, and the timing of reproductive events in an Asian primate, the Hanuman langur (Presbytis entellus). J Zool (London) 1997; 243:215–35. [4] Bronson FH, Macmillan B. Hormonal responses to primer pheromones. In: Vandenbergh JG, editor. Pheromones and reproduction in mammals. New York: Academic Press; 1983. p. 175–97. [5] Signoret JP. Sexual pheromones in the domestic sheep: importance and limits in the regulation of reproductive physiology. J Steroid Biochem Mol Biol 1991;39:639–45. [6] Hastings MH, Herbert J, Martensz ND, Roberts AC. Annual reproductive rhythms in mammals: mechanism of light synchronization. Ann NY Acad Sci 1985;453:182–204. [7] Di Bitetti MS, Janson CH. When will the stork arrive? Patterns of birth seasonality in neotropical primates. Am J Primatol 2000; 50:109–30. [8] Anderson DP, Nordheim EV, Boesch C. Environmental factors influencing the seasonality of estrus in chimpanzees. Primates 2006;47:43–50. [9] Bicca-Marques JC, Fichtner D. Birth seasonality of Cebus paella (Platyrrhini, Cebidae) in Brazilian Zoos along a latitudinal gradient. Am J Primatol 2005;65:141–7. [10] Strier KB, Mendes SL, Santos RR. Timing of births in sympatric brown howler monkeys (Alouatta fusca clamitans) and northern muriquis (Brachyteles arachnoides hypoxanthus). Am J Primatol 2001;55:87–100. [11] Milton K. Estimates of reproductive parameters for free-ranging Ateles geoffroyi. Primates 1981;22:574–9. [12] Fedigan LM, Rose LM. Interbirth interval variation in three sympatric species of neotropical monkey. Am J Primatol 1995; 37:9–24.
1993
[13] Klein LL. Observations on copulations and seasonal reproduction of two species of spider monkeys, Ateles belzebuth and A. geoffroyi. Fol Primatol 1971;15:233–48. [14] Chapman C, Chapman L. Reproductive biology of captive and free-ranging spider monkeys. Zoo Biol 1990;9:1–9. [15] Herna´ndez-Lo´pez L, Mayagoitia L, Esquivel-Lacroix C, RojasMaya S, Mondrago´n-Ceballos R. The menstrual cycle of the spider monkey (Ateles geoffroyi). Am J Primatol 1998;44:183– 95. [16] Herna´ndez-Lo´pez L, Cerezo-Parra G, Cerda-Molina AL, Pe´rezBolan˜os SC, Dı´az-Sa´nchez V, Mondrago´n-Ceballos R. Sperm quality differences between the rainy and dry seasons in captive black-handed spider monkeys (Ateles geoffroyi). Am J Primatol 2002;57:35–41. [17] Matsumuro M, Tadashi S, Fumiaki C, Yasuhiro Y, Takashi Y. A two-step extraction method to measure fecal hormones in female cynomulgus monkey (Macaca fascicularis). Am J Primatol 1999;48:291–8. [18] Ziegler T, Hodges K, Winkler P, Heistermann M. Hormonal correlates of reproductive seasonality in wild female hanuman langurs (Presbytis entellus). Am J Primatol 2000;51: 119–34. [19] Strier KB, Ziegler TE. Behavioral and endocrine characteristics of the reproductive cycle in wild muriqui monkeys, Brachyteles arachnoids. Am J Primatol 1997;42:299–310. [20] Campbell C, Shideler SE, Todd HE, Lasley BL. Fecal analysis of ovarian cycles in female black-handed spider monkeys (Ateles geoffroyi). Am J Primatol 2001;54:79–89. [21] Campbell C. Patterns of behavior across reproductive states of free-ranging female black-handed spider monkeys (Ateles geoffroyi). Am J Phys Anthropol 2004;124:166–76. [22] Dixson AF, Primate Sexuality. Comparative studies of the prosimians, monkeys, apes and human beings Oxford: Oxford University Press; 1998. p. 444–53. [23] Eisenberg JF. Communication mechanism and social integration in the black spider monkey, Ateles fusciceps robustus, and related species. Smithson Contrib Zool 1976;213:1–108. [24] Van Roosmalen MG, M Klein L. The spider monkeys, genus Ateles. In: Mittermeier R, Rylands A, editors. Ecology and behavior of neotropical primates. Washington, DC: World Wildlife Fund; 1988. p. 455–537. [25] Pe´rez-Ruiz AL. Disponibilidad de alimento y comportamiento afiliativo del mono aran˜a (Ateles geoffroyi) en la selva lacandona, Chiapas. Master Degree Thesis; 2004. [26] Estrada A, Coates-Estrada R. Some observations on the present distribution and conservation of Alouatta and Ateles in southern Mexico. Am J Primatol 1984;7:133–7. [27] Goldizen AW, Tergorgh J, Cornejo F, Porras DT, Evans R. Seasonal food shortage, weight loss, and the timing of births in saddle-back tamarins (Saguinus fuscicollis). J Anim Ecol 1988;57:893–901.