Assessing reproductive profiles in female brown mouse lemurs (Microcebus rufus) from Ranomafana National Park southeast Madagascar using fecal hormone analysis.код для вставкиСкачать
American Journal of Primatology 71:439–446 (2009) RESEARCH ARTICLE Assessing Reproductive Profiles in Female Brown Mouse Lemurs (Microcebus rufus) From Ranomafana National Park, Southeast Madagascar, Using Fecal Hormone Analysis MARINA B. BLANCO1 AND JERROLD S. MEYER2 1 Department of Anthropology, University of Massachusetts, Amherst, Massachusetts 2 Department of Psychology, University of Massachusetts, Amherst, Massachusetts Studies on reproductive endocrinology in wild primate populations have greatly increased in the last decades owing to the development of noninvasive techniques that can be applied under field conditions. However, small-bodied nocturnal species are not well represented on the long list of primates surveyed in the wild, and reproductive inferences regarding these animals in their natural habitats have not benefited from direct observations of hormonal changes. We collected fecal samples from female brown mouse lemurs (Microcebus rufus) in a southeastern rainforest of Madagascar in order to determine whether or not fecally excreted steroid levels show a consistent pattern of change during the reproductive season and are a useful complement to reproductive observations in wild-trapped individuals. Initial data show variation in reproductive hormone levels before and after estrus and estimated day of parturition. Elevated levels of excreted estradiol (E2) were observed around the time of estrus, whereas high levels of fecal progesterone (P) were seen during later stages of pregnancy and around parturition. A more complete picture of reproductive profiles in female mouse lemurs, and how they may change over the life span, can be obtained if hormone analyses are used to supplement field observations. Am. J. Primatol. 71:439–446, 2009. r 2009 Wiley-Liss, Inc. Key words: Microcebus rufus; Ranomafana National Park; estradiol; progesterone; fecal analysis INTRODUCTION Noninvasive endocrine analyses of ovarian function have been applied successfully to a variety of captive as well as wild primate populations. Ever since the pioneering work by Risler et al.  and Wasser et al. , different methodologies have been explored and validated under captive conditions, where the reproductive status of individuals can be regularly monitored and the relative efficacy of different types of samples (e.g. urine, blood, feces) can be assessed. The consensus that fecal steroids are informative vis-à-vis reproductive function has greatly influenced the field of primate endocrinology, and it has facilitated the analyses of endangered species for which only noninvasive techniques can be applied [Strier & Ziegler, 1997]. To date, fecal endocrine profiles that employ a single or a combination of reproductive hormones have been reported for many primate groups. These include sifakas, red fronted lemurs and mongoose lemurs among lemuroids [Brockman & Whitten, 1996; Brockman et al., 1995; Curtis et al., 2000; Ostner & Heistermann, 2003]; tamarins, marmosets, muriquis and saki monkeys among ceboids [Heistermann et al., 1993; Shideler et al., 1994; Strier et al., 2003; Ziegler et al., 1996]; a variety of macaques and langurs among r 2009 Wiley-Liss, Inc. cercopithecoids [Bardi et al., 2003; Heistermann et al., 1995; Shideler et al., 1993]; and bonobos and gorillas among hominoids [Atsalis & Margulis, 2006; Jurke et al., 2002; Miyamoto et al., 2001]. Some groups, however, are poorly represented or entirely missing from the list. In particular, there is a paucity of data on small-bodied nocturnal lemurs in the wild. This bias toward larger diurnal primates may be in part logistical. It is difficult to collect fecal samples during focal animal observations at night when the animals are active; thus, feces are most easily gathered when individuals are captured in traps. Frequent capture and recapture of the same individual may be required to obtain sufficient samples to reconstruct its endocrine profile, a task Contract grant sponsors: Rufford Foundation; MMBF/CI Primate Action Fund; Institute of Biotechnology; NSF; Contract grant number: BCS-0721233. Correspondence to: Marina B. Blanco, Department of Anthropology, 240 Hicks Way, University of Massachusetts, Amherst, MA 01003. E-mail: firstname.lastname@example.org Received 15 March 2008; revised 8 December 2008; revision accepted 18 January 2009 DOI 10.1002/ajp.20672 Published online 10 February 2009 in Wiley InterScience (www. interscience.wiley.com). 440 / Blanco and Meyer that cannot be guaranteed (i.e. other individuals may enter the same traps at any given night) and may indeed disrupt reproductive schedules if the same individual enter the same traps every single day. Our study species, the brown mouse lemur, Microcebus rufus, is a small-bodied (40 g) nocturnal seasonal breeder that inhabits the eastern rainforests of Madagascar. Although brown mouse lemurs as well as gray mouse lemurs (M. murinus), a western species, have been kept in captivity for decades [Glatston, 1979; Perret, 1986; Wrogemann & Zimmermann, 2001], little has been published on their endocrine profiles. Furthermore, there is currently a lack of consensus regarding the utility of fecal analysis and even less understanding of individual variability in mouse lemur steroid profiles, and particularly how such profiles may change, over their life span. Glatston  conducted preliminary hormone analysis in captive female mouse lemurs in an attempt to detect the early stages of pregnancy, but no urinary estrogen peaks were detected during estrus or later. Further studies on reproductive hormones have shown mixed results. In 1986, Perret reported consistent plasma progesterone (P) levels in consecutive estrous cycles of captive M. murinus females. Variation in hormonal levels among individuals was attributed to different social conditions. A subsequent study of the same species found, but could not explain, high intra- and inter-individual variation in serum P [Buesching et al., 1998]. The latter study also attempted to use urinary and fecal estrogen and P metabolites to monitor reproductive function noninvasively; however, no clear pattern of change in steroid levels within the reproductive cycle was found. Finally, Perret  showed a clear progressive increase of urinary estradiol (E2) 10 days before estrus, a peak of excretion at ovulation, and a consequent decrease to baseline levels within 4 days, although she reported significant variation among individual females, and she did not test fecal steroids. Collecting serum is highly invasive and collecting urine from wild-caught animals is logistically difficult given the problems posed by preservation and storage of urine under field conditions. Preservation of dried-oven fecal samples is easier, and it seems possible that prior apparent negative results may be owing to low assay sensitivity or hormonal variability over the life span. This in itself may be of biological interest. Because so little is known about fecal steroids in mouse lemurs even in captivity, sampling fecal steroids should not be abandoned without more rigorous testing. We now report data obtained from live-trapped mouse lemurs at Ranomafana National Park, a southeastern rain forest in Madagascar. Our aims are to (1) determine whether or not a clear pattern of fecal E2 and P excretion exists in female brown mouse lemurs during the Am. J. Primatol. reproductive season; (2) document the efficacy of minimally invasive fecal hormonal analysis as a reliable marker of ovulation, parturition and early lactation, within the context of overall individual variability and (3) explore factors (such as age) that might help us to understand the pattern of variation among individuals. This study represents the first report on fecal steroid analysis as a tool to monitor reproductive function in wild mouse lemurs. METHODS This research, conducted under permission of institutional and governmental agencies that regulate animal research in Madagascar, adhered to the American Society of Primatologists Principles for the Ethical Treatment of Nonhuman Primates. Research protocols complied with those approved by the University of Massachusetts—Amherst Animal Care and Use Committee. Study Area and Field Observations We conducted field work in the Talatakely Trail System at Ranomafana National Park, a montane rainforest in southeast Madagascar (471180 –471370 E and 211020 –211250 S). We employed capture/mark/ recapture techniques in October–December 2005, October 2006–January 2007 and October 2007 as a part of a long-term monitoring of mouse lemur populations in this study area (9 ha). A maximum of 50 Sherman traps at intervals of ca. 25 m were baited with a piece of fresh banana and set up daily between 16:00 and 17:00 hr along preexisting trails (similar areas have been surveyed over the years to maximize recapture rates) on consecutive nights (trapping nights: 69 in 2005; 54 in 2006, 21 in 2007). Traps were checked between 19:30 and 21:00 hr, and trapped animals were brought to the ValBio research station where individuals were identified or marked with Avid microchips, weighed and handled to observe their reproductive status. Trapped Microcebus were released between 23:00 and 1:00 hr on the same night of capture to minimize disturbance of their reproductive schedules. Reproductive status of females was determined by inspecting vaginal morphology; i.e. diestrous condition was characterized by sealed and swollen vaginas; proestrous, estrous and metestrous conditions were determined during vaginal openings through the inspection of cytological features at 40 magnification from vaginal smears. Physiological estrus was determined by the presence of sperm (decapitated heads and/or tails), vaginal plugs or by the inspection of cytological features from the smears following descriptions for mouse lemurs available in the literature [e.g. Blanco, 2008; Wrogemann & Zimmermann, 2001]; additional observations such as nipple development and body mass were used to generally distinguish the later stages of pregnancy and early Fecal Excreted Steroids in Microcebus rufus / 441 lactation. More information about nipple development and body mass changes during gestation is provided in Blanco . Gestation length could be established with confidence in three cases (‘‘I’’, ‘‘J’’ and ‘‘K’’ in 2005) giving a period of 57 days [Blanco, 2008]. These estimations agree with published records from captivity [Wrogemann & Zimmermann, 2001]; the date of parturition of the remaining females was then estimated by adding 57 days to the date of estrus. We could preliminarily classify the female population into two broad age categories: young (1–2 years old) vs. adults (Z3 years old) based on the pattern of dental wear and body mass. Zodhy et al. [personal communication] have developed an age scale based on digital measurements from high-quality dental molds from frequently captured individuals. In addition, 1-year-old individuals tend to be lighter than older females at the beginning of the reproductive season [personal observation]. We collected fecal samples from animals that defecated when handled. If fresh feces were not available, then we recovered samples from inside the Sherman traps where the individual females had remained for a period no longer than 3 hr. Feces were weighed with a digital balance (accurate to 0.01 g), wrapped in aluminum foil, and oven-dried for about 2–3 days at 501C. Fecal pellets were then stored in plastic bags at room temperature until the end of the field season (3.5-month period). Once at the University of Massachusetts—Amherst, samples were kept frozen at 201C until assayed. Except for female ‘‘V’’ (for which samples collected in 2005 were assayed in 2007), all fecal samples were analyzed within 6 months after being brought back from the field. We used SPSS 15.0 to calculate statistical significance (a 5 0.05) in parametric (Independent T-tests, Levene’s Tests for Equality of Variance) and nonparametric tests (Sign Test). One-tailed tests of significance were used to test specific directional hypotheses—for example, that E2 should decrease following ovulation (H0 5 there is no change in E2 over ovulation, or E2 increases following ovulation). Fecal Sample Preparation The protocol that we employed was modified from that of Khan et al. . Fecal pellets were ground with a Mixer Mill MM200 milling machine (Retsch Inc., Newtown, PA). This method produces a finer powder than would be obtained by hand grinding, which should improve hormone extraction from small samples. After milling, the samples were weighed and transferred to 2 ml microcentrifuge tubes containing an extraction solution consisting of 1.5 ml 90% methanol and 50 ml of [3H]E2 (approximately 75,000 CPM; 83.0 Ci/mmol, Amersham Biosciences, PA) in ethanol as an internal standard. For later determination of steroid recovery, 300 ml of the same extraction solution were added to 10 ml of scintillation cocktail (ScintisafeTMSX 23–5, Fisher Scientific, PA) in duplicate and radioactivity was measured in a Packard 1900CA scintillation counter. Samples were extracted overnight at room temperature using a rotator. The following morning, samples were centrifuged for 10 min (speed 5, Marathon Micro A, Fisher Scientific) and 800 ml of the supernatant was transferred into a clean glass tube and dried under nitrogen gas. Samples were reconstituted in 400 ml of 30% methanol and vortexed intensively before running the solid-phase extraction. Fecal steroids were extracted using solid-phase Oasis cartridges (3cc/60 mg HLB, Waters, Milford, MA). Cartridges were conditioned with 1.0 ml of 100% methanol followed by 1.0 ml of deionized water. Each cartridge was loaded with 300 ml of each sample and rinsed with 1.0 ml of 20% methanol. Steroids were eluted from the columns with 2.0 ml of 100% methanol [Khan et al., 2002]. Samples were stored at 201C until assayed. Steroid Radioimmunoassays (RIAs) Before conducting steroid RIAs, samples were dried under nitrogen gas and reconstituted with 300 ml of 30% methanol. One hundred microliter of each sample were added to 10 ml of the scintillation cocktail and radioactivity was measured to determine steroid recovery. For the E2 assay, 15 ml of each sample was diluted in 1.485 ml of the diluent provided in the RIA kit before being assayed. To measure fecal E2 we employed the Pantex 125I Estradiol kit (catalog ]047, Santa Barbara, CA) and followed the manufacturer’s instructions. Crossreactivity provided by the manufacturer is 1.4% for a-estradiol and less than 0.02% for other steroids. Our estimated intra-assay Coefficient of Variation (CV) for E2 was 11%. Briefly, serial dilutions of a calibrated standard and the fecal extracts at a volume of 500 ml were assayed in duplicate. One hundred microliter of tracer was added to each tube, followed by 100 ml of the first antiserum. After 30 min of incubation at 371C, 500 ml of the second antiserum was added to the samples and the standards. Tubes were vortexed, left at room temperature for 10 min, and then centrifuged at 2400 rpm for 15 min. Supernatants were discarded immediately and radioactivity of precipitates was counted using a Packard gamma counter (CobraTM II, Packard Instrument Company, CI). To assess levels of fecal P, we used the Pantex 125I Direct Progesterone kit (catalog ]137, Santa Barbara, CA), which is highly specific for P with cross-reactivity of 0.5% for hydroxyprogesterone, 0.1% for androsterone and values below 0.1% for other steroids. Intra-assay CV for P was 10.9%. For the P assay, 50 ml of each sample was diluted in 450 ml of diluent. The standards and the samples (100 ml) were prepared in duplicate and 500 ml of tracer were Am. J. Primatol. 442 / Blanco and Meyer added to the tubes, followed by 100 ml of the first antiserum. Tubes were vortexed and incubated at 371C for 1 hr. Then 500 ml of the second antiserum was added and tubes were left at room temperature for 10 min. Finally, the tubes were centrifuged at 2400 rpm for 15 min, supernatants were immediately discarded, and precipitate radioactivity was counted as before. RESULTS A total of 47 individual females were captured during the reproductive seasons of 2005, 2006 and 2007. We determined physiological estrus from vaginal smears (n 5 17) and estimated dates of parturition for 10 individual females trapped during the study period. One hundred twenty fecal samples were assayed for excreted E2 and P. Females ‘‘V’’, ‘‘Ke’’ and ‘‘S’’ contributed fecal samples for two consecutive years and ‘‘I’’ and ‘‘J’’ for all three seasons. Hormone levels, expressed as pg/mg for E2 and ng/mg for P, were corrected for steroid recovery in each sample, which averaged 73% in samples assayed in 2008, and 76 and 77% in samples analyzed in 2007 and 2006, respectively. In all, seven females were sampled around the time of estrus for E2 (for a total of 11 cases) and six females were sampled for P during the same period (for a total of ten cases). Around the estimated day of parturition, samples from nine females were available for E2 (for a total of 18 cases) and P (for a total of 16 cases). In nine of 11 cases, E2 levels were higher before or at estrus (day 10 to day 0) than during the 5-day period after estrus (day11 to day15). This difference was statistically significant (Sign Test, one-tailed, P 5 0.03, Fig. 1). There was substantial intra- and inter-individual variation in E2, as illustrated by maximum pre- and post-estrus E2 levels in Table I. Variation was significantly higher in young than in adult individuals (Table II), although the smaller number of young animals may have contributed to this difference. In six of ten cases, P levels increased after estrus (day11 to 15) but generally remained relatively low. The change from pre- to post-estrus in the sample was not statistically significant (Sign Test, one-tailed, P 5 0.37, Fig. 2). Figure 3 illustrates the hormonal profile of individual ‘‘I’’ in 2006, which is consistent with changes in vaginal morphology around the time of estrus. Owing to a lack of certainty concerning the exact day of parturition for some of the pregnant females, we designated the prepartum period to be 10 to 0 (71) days. E2 levels tended to increase during this period followed by a decrease after parturition. Although the post-partum pattern is clearly shown in Figure 4, no significant differences were found, possibly owing to small sample sizes (particularly the few number of animals for which samples were available for all three periods) as well as the high intra- and inter-individual variation (Table IIIa). Similarly, P levels were higher during the later stages of pregnancy than during the weeks following parturition (Fig. 5; Table IIIb). Fig. 1. E2 levels before (10 to 0 days) and after estrus (11 to 15 days). Each symbol represents an individual’s mean values within that period. One-tailed Sign Test, P 5 0.03. TABLE I. Maximum E2 Values Before and After Estrus for Young (1–2-year-old) and Adult (Z3-year-old) Females Femalea K 05 S 06 S 07 V 06 A 07 I 05 I 06 I 07 J 07 Ke 06 Ke 07 a Est. age E2 Days from estrus E2 Days after estrus Young Young Young Young Adult Adult Adult Adult Adult Adult Adult 163.2 74.5 655.9 33.7 28.6 118.3 160.5 138.6 89.7 30.9 48.2 0 1 4 2 0 2 4 5 0 4 0 16.7 156.1 51.0 142.3 14.8 21.6 16.8 30.8 23.6 12.0 6.7 3 4 2 1 5 2 5 1 2 2 1 Females are identified by initials and year of capture. Am. J. Primatol. Fecal Excreted Steroids in Microcebus rufus / 443 TABLE II. Mean E2 Values Prior to (10 to 0 days) and After Estrus (11 to 15 days) for Young and Adult Females Pre-estrus Young (n 5 4) Adults (n 5 7) Post-estrus Mean SDa Mean SDb 106.9 48.8 62.8 22.3 88.5 17.2 70.9 7.9 a F 5 22.9, P 5 0.001. F 5 140, Po0.001 by Levene’s Test for equality of variance. Each mean is the average of the mean values for each of the individuals in the sample. b Fig. 4. E2 levels before and after estimated parturition (7 1day). Means were used for individual females with multiple values within each period. TABLE III. Descriptive Statistics for (a) E2 and (b) P Before and After the Estimated day of Parturition (71 day) Time period (a) Estradiol (E2) [–10 to 10]1 [12 to 114]2 [115 to 131]3 Fig. 2. P levels before (10 to 0 days) and after estrus (11 to 15 days). Each symbol represents an individual’s mean values within that period. One-tailed Sign Test, P 5 0.38. Fig. 3. Hormone profile of female I 06 around the time of estrus. E2 peaked before vaginal opening and P began to rise at the end of the first week of pregnancy. Reproductive observations: 7: no vaginal opening; 4: swollen vagina; 2: very swollen vagina; 0: vaginal plug; 15: vaginal opening, but in the process of closing. N Mean SD 9 6 5 74.4 35.6 42.4 45.8 35.6 50.4 8 5 5 2.4 1.2 1.1 1.2 0.9 0.6 a (b) Progesterone (P)b [10 to 10]1 [12 to 114]2 [115 to 131]3 a Independent sample t-test for equivalence of means: 1,2t 5 1.7, df 5 13, P 5 0.1; 2,3t 5 0.3, df 5 9; P 5 0.8; 1,3t 5 1.2, df 5 12, P 5 0.25. b Independent sample t-test for equivalence of means: 1,2t 5 2.0, df 5 11, P 5 0.07; 2,3t 5 0.1, df 5 8, P 5 0.9; 1,3 t 5 2.3, df 5 11, P 5 0.04. Fig. 5. P levels before and after estimated parturition (7 1 day). Means were used for individual females with multiple values within each period. Am. J. Primatol. 444 / Blanco and Meyer DISCUSSION Our data indicate that successful extraction of female reproductive hormones can be obtained from oven-dried fecal pellets collected in the field from small live-trapped nocturnal lemurs. Lynch et al.  pointed out that fecal data are ‘‘noisier’’ than serum or urine data, which is likely owing to variation in the composition of fecal matter and bacterial degradation among other factors. On the other hand, fecal collection obviates the need for more invasive procedures such as blood withdrawal that could result in stress-induced variation. Furthermore, fecal samples contain analytes that have accumulated between defecation events, thus potentially providing a more representative ‘‘picture’’ of the reproductive state of an individual. In contrast, values obtained from other sources such as serum represent ‘‘snapshots’’ of metabolic and chemical levels at a given point in time [Bardi et al., 2003; Whitten et al., 1998]. Our data show a pattern of hormonal variation that is generally consistent with expectations derived from published data in other lemur species. Fecal E2 levels increased before or at the day of estrus, and subsequently decreased, in agreement with results from urinary E2 measurements in gray mouse lemurs reported by Perret . During the final 10 days before parturition, however, fecal E2 levels were higher than post-estrus values. This finding is consistent with results from some other lemuroids, such as captive sifakas (Propithecus verreauxi) [Brockman et al., 1995], but not wild mongoose lemurs (Eulemur mongoz) [Curtis et al., 2000]. Elevated levels of E2 shortly before parturition may stimulate lactogenesis by indirectly enhancing prolactin secretion by the pituitary gland [Hadley, 2000]. However, E2 levels should be interpreted with caution. Ostner and Heistermann  suggested that variation in fecal estrogen excretion during the second half of pregnancy in female red fronted lemurs (E. fulvus rufus) is related to the sex of the offspring, with increased estrogen levels occurring only in females giving birth to males. This may be a prosimian characteristic, as this phenomenon has been also reported in ruffed lemurs (Varecia variegata) and tentatively mongoose lemurs as well [Ostner & Heistermann, 2003]. We also found that fecal P levels started to rise after estrus in most of the females, although values were consistently low during ovulation and early pregnancy (Fig. 2). Similarly, Buesching et al.  observed that vaginal opening in captive female gray mouse lemurs occurred when plasma P levels reached their minimum and considered this hormone to be an unreliable marker of reproductive state at the time of estrus. Perret  attributed variation of plasma P levels during the luteal phase of nonpregnant females to differences in social Am. J. Primatol. conditions. We observed higher P values around the time of parturition (Fig. 3). Those levels started to decrease after birth, although they remained elevated above luteal values for at least 2 weeks, a considerably longer period than that reported for red fronted lemurs in which low mating concentrations of fecal progestins were attained within 5 days after parturition [Ostner & Heistermann, 2003]. It has been reported that post-partum secretion of P in females with suckling infants is owing to the maintenance of the corpus luteum of pregnancy via increased levels of prolactin associated with lactation [Knobil & Neill, 2006]. Finally, we observed significant intra- and interindividual variation in both E2 and P throughout the reproductive season. This may hinder the use of fecal hormonal analysis as a reliable tool to determine reproductive condition in the absence of other supplementary data (e.g. observations of vaginal morphology, nipple development, abdomen palpation or behavioral observations). We suggest, however, that the pattern of variation may be worth studying. Buesching et al.  and Perret  observed variability in hormone levels in their studies on gray mouse lemurs in captivity. Perret  reported relatively higher and variable urinary E2 levels in younger individuals at estrus and higher E2 levels before estrus in female mouse lemurs giving birth to female-biased litters. Although it is not possible to test the latter observation under these study conditions, we did find, in agreement with Perret, more variation in E2 levels around estrus in the younger females (Table II). Another source of variation in P is spontaneous resorptions during early pregnancies that may pass undetected without direct hormonal sampling. Although Perret  suggested that early undetected resorptions can shorten interestrous intervals in mouse lemur females, further hormonal analyses are necessary to corroborate this proposition. Even perinatal death of the offspring would trigger the physiological changes associated with a renewed estrus (e.g. abrupt decreases of P during apparent pregnancy or right after parturition). In turn, both instances may be correlated with age, if younger individuals, for example, are prone to reproductive failures more often than adults. We report the case of female ‘‘K’’, who was a young female in 2005 (1-year-old) and was frequently captured during the reproductive season. She showed high E2 levels on the day of estrus (Table I), but experienced an irregular weight gain profile during pregnancy [personal observation]. Furthermore, she displayed a swollen vagina on the last day of capture (December 22, 2005, about 5 days after estimated parturition), suggesting that her offspring, if born alive, had probably died. Her samples showed relatively low E2 levels before parturition (Fig. 4) and a sharp decrease in P after giving birth (values were below the sensitivity of the Fecal Excreted Steroids in Microcebus rufus / 445 assay). Thus, her hormone ‘‘profile’’ in addition to body mass data and observations of vaginal morphology provide a more complete picture of her reproductive status. Such findings support the contention that fecal hormonal analysis can be a useful supplementary tool to help understand the pattern of reproductive variation in wild primate populations. In conclusion, this study represents the first report of fecal E2 and P levels from wild nocturnal lemurs as well as the first to use a milling machine to grind fecal pellets for steroid extraction in a primate. Fecal E2 and P levels obtained from wild brown mouse lemur females during the reproductive season were generally consistent with cytological and captive data, but intra- and inter-individual variation was found to be significant and widespread during the reproductive season. The application of less-invasive techniques to construct endocrine profiles in small-bodied nocturnal lemurs in the wild has the potential to increase our knowledge of their reproductive biology by providing additional data that may not be available through direct observations of wild-trapped individuals. Beyond the analysis of individual hormonal profiles, investigation of possible sources of variation at the population level, such as age and litter characteristics (e.g. sex and size), may enhance our understanding of reproduction and population dynamics of nocturnal lemurs in the wild. ACKNOWLEDGMENTS This project benefited from the help of many people, particularly Laurie Godfrey and Lynnette Sievert, who provided advice at every stage. We are thankful to Anja Deppe for her collaboration with mouse lemur research as well as Patricia Wright for support in the field. Additional thanks go to our local (Centre ValBio-trained) research assistant Victor Rasendrinirina, the Centre ValBio director Anna Feistner, Jean Claude Razafimahaimodison, Aimée Razafiarimalala and other personnel of the Centre ValBio for logistic support. Fieldwork in Madagascar was conducted under permission of the Ministry of Environment, Water and Forests, and the CAFF/ CORE committee and our research protocol was authorized by the University of Massachusetts IACUC. Research in Madagascar was facilitated by staff from the Association Nationale pour la Gestion des Aires Protégées (ANGAP), the Institute for the Conservation of Tropical Environments (ICTE, Stony Brook) and the Madagascar Institute pour la Conservation des Ecosystèmes Tropicaux, (MICET), especially its director, Benjamin Andriamihaja. We thank Jeff Wyatt for veterinary advice. Comments on an earlier draft of this manuscript by Nancy Forger, Sylvia Atsalis, Toni Ziegler and Anna Feistner are also greatly appreciated. This paper was written with the support of the Rufford Foundation, MMBF/CI Primate Action Fund and Institute of Biotechnology, Helsinki to M. B. B. and NSF BCS-0721233 to Patricia C. Wright, Laurie R. Godfrey and Jukka Jernvall. Comments from two anonymous reviewers on previous versions of this manuscript are greatly appreciated. REFERENCES Atsalis S, Margulis SW. 2006. Sexual and hormonal cycles in geriatric Gorilla gorilla gorilla. Int J Primatol 27: 1663–1687. 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