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Gholib Assahad, PhD student, German Primate Center, Bogor Agricultural University - 2017 completed

Crested macaque is one of the endemic Sulawesi macaques  which can only be found at the tip of North Sulawesi island, Indonesia. The population of this species having declined and has been categorized as critically endangered species. With the rapidly declining populations, efforts to protect and increase the population of crested macaques is becoming increasingly important. Therefore, monitoring of reproduction and stress are needed. The aim of the present study was to determine the pregnancy, parturition , and monitoring  stress non invasively based on the fecal hormone analyses and observation of  the sexual signal, sex skin coloration. This study  include the following steps: (1) validation of enzyme immunoassays for measuring steroid metabolites (progesterone, estrogen, and glucocorticoid); (2) validation of  fecal preservation, extraction, and storage method; (3) determination of the pregnancy and parturition through observation of sex skin coloration  and fecal hormone analysis; and (4) measurement of stress hormone in females during cycling, pregnancy, and lactation. This study was conducted at Tangkoko-Batuangus Nature Reserve, North Sulawesi (1⁰33' N, 125⁰10' E), a natural habitat of wild crested macaques. In total 12 females from a group of Rambo I were observed during this study.

The results of the first stage of the study: levels of estrogen metabolite (fEM) were lower at the beginning of the follicular phase and increased at the end of the folliculer phase (ovulation), subsequently decreased again in the luteal phase (p <0.05). From three  of estrogen hormone assays tested, E1C showed a better profile compared to  the two other  hormone assays (estradiol, and Etotal). The three progesterone hormone assays (PdG, progesterone, and pregnanolone) tested showed that levels of progesterone metabolite (fPM) in the follicular phase were lower compared to the levels of fPM during the luteal phase (p<0.05). However, the baseline of pregnanolone hormone assay in the follicular phase was  more stable than the two other hormone assays. The three glucocorticoid hormone assays validated (cortisol, 11-oxo-CM, and 11β-hydroxy-CM) showed that glucocorticoid levels in all assays increased at 2-3 days after stress events occured. However, in the 11β-hydroxy-CM assay, glucocorticoid levels were higher compared to 11-oxo-CM and cortisol (p <0.05).

Results of the second  stage of the study: levels of fEM were higher, while levels of glucocorticoid metabolite (GCM) were lower in feces preserved 10 hours after collection compared to control (feces stored immediately at -20OC;       p<0.05). Long-term storage of feces in a 80% methanol (MeOH) led to a marked increase in the levels of fEM and GCM after the 4th and 6th months of storage, respectively (p<0.05). Drying the feces using the oven-drying method at 50°C, 70°C, and 90°C did not affect the fEM levels (p> 0.05), but drying at 90°C caused a significant lower of GCM levels compared to 50OC, and 70OC (p<0.05). Three different fecal preservation methods (freeze-drying , field frienly extraction, and oven drying) and extraction solvents (MeOH, ethanol/EtOH, and commercial alcohols) resulted similar levels of fEM and GCM (P>0.05). Freeze-thaw cycles in fecal extracts caused  a significant increase of fEM levels after 8 times of freeze-thaw cycles and 4 time of freeze-thaw cycles in GCM (p<0.05).

The results of the third stage of the study: the color intensity of sex skin coloration changed in the third and fourth weeks after the detumescent which it becomes light pink with the value of R/G (red/green) ratio = 1.67±0.12 if the female was not pregnant and it becomes red  with the value of R/G ratio = 1.94±0.16 if the female was pregnant. The intensity of the red color becomes dark red in line with the age of gestation and becomes magenta with the value of R/B (red/blue) ratio = 0.28±0,11 one week prior to parturation. The changes in color intensity were in line with the increase the levels of fEM in which the fEM levels begins to increase at the fourth week of gestation (118.90±16,90 ng/g) higher compared to the baseline of fEM levels during follicular phase (78.8±5.64 ng/g  and continue to increase until few days prior to parturation. Glucocorticoid levels at the first trimester of gestation (403.38±62.75 ng/g) and the second trimester of gestation (596.41±107.80 ng/g) were lower than in the third trimester of gestation (1086.23±125.66 ng/g), and increased very sharply a week prior to parturition (1911.84±171.22 ng/g).

The results of the fourth stage of the study, levels of glucocorticoid in pregnant females were significantly higher (814.20±139.1 ng/g) compared to the levels of  glucocorticoid in females during cycling and lactation 388.19±60.59 ng/g, and 403.13±39.34 ng/g, respectively (p<0.05). This is likely caused by  the increase of  metabolic demands  and the interactions between the HPA axis, estrogens, and placental production of corticotrophin releasing hormones, then released the glucocorticoids.

Based on the results, assessing pregnancy, parturion, and monitoring stress in crested macaques can be performed non-invasively through the observation of sex skin coloration and hormone analysis. The non-invasive approach was more suitable for study in wild animals living in their natural habitas.  This finding provided  an important contribution directly or indirectly towards the breeding program and conservation in this species.

Keywords: crested macaques, sex skin coloration, pregnancy, parturition, stress