Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

  • Print ISSN 0367-6722

  • Online ISSN 0976-0555

  • NAAS Rating 6.50

  • SJR 0.263

  • Impact Factor 0.4 (2024)

Frequency :
Monthly (January, February, March, April, May, June, July, August, September, October, November and December)
Indexing Services :
Science Citation Index Expanded, BIOSIS Preview, ISI Citation Index, Biological Abstracts, Scopus, AGRICOLA, Google Scholar, CrossRef, CAB Abstracting Journals, Chemical Abstracts, Indian Science Abstracts, EBSCO Indexing Services, Index Copernicus

A Proposed Mating Guideline for Bornean Orangutans (Pongo pygmaeus) using Urinary Hormonal Tracking Methods

Liang-Jun Tseng1, Jane-Fang Yu2, Yu-Chia Chang2, Lih-Chiann Wang1,*
1The School of Veterinary Medicine, National Taiwan University, No 1, Sec 4, Roosevelt Rd, Taipei 10617, Taiwan.
2Taipei Zoo, No 30, Sec 2, Xinguang Rd, Taipei 11656, Taiwan.
Bornean orangutans (Pongo pygmaeus) are a critically endangered species. Proper monitoring of the menstrual cycle is important in breeding programs. Hormonal cycle tracking was performed in this study by collecting female urine. Metabolites of estrogen (estrone glucuronide, E1g) progesterone (P4) were measured using enzyme immunoassay. E1g tracking was carried out for 8 animal-months during normal cycling to obtain an average hormonal profile. P4 was tracked throughout normal cycling to obtain a baseline and over 1 successful pregnancy to obtain a reference for pregnancy monitoring and parturition anticipation. This study concludes that this non-invasive hormonal cycle monitoring using urine is an acceptable method. A recommended mating guideline of introducing the male after an estrogen increase of 1.5 times the baseline is proposed based on the results. This minimizes coerced mating and increases chances of pregnancy, which would benefit animal welfare and greatly assist in the conservation of the species.
Bornean orangutan (Pongo pygmaeus) is listed as a critically endangered species (Ancrenaz et al., 2016). Conservation of these species is of upmost importance. E1g tracking is essential to accurately estimate the ovulation timing and assist in breeding. P4 levels can also be used as a marker to confirm a pregnancy and initiate proper pregnancy preparation care to the expectant female (Noakes et al., 2009). Some male orangutans have been known to engage in forceful copulation if kept with a female that is not in estrus, which may cause injury to the female. In such cases, it would be advantageous to house the aggressive males away from the females and only introduce them during periods of estrus. Ovulation tracking allows safe contact consensual mating (Atmoko et al., 2009; Nadler, 1995). Non-invasive methods using fecal or urinary samples to test for the hormone metabolites is preferable in animals (Hodges Heistermann, 2011; Knott, 1996). This process has already been studied in humans (Munro et al., 1991) other primates, including the mountain gorilla (Gorilla beringei beringei), chimpanzees (Pan troglodytes) and bonobo (Pan paniscus) (Habumuremyi et al., 2014; Kinoshita et al., 2016; Shimizu et al., 2003). However, even among closely related species, hormonal metabolites found in their excretions may vary greatly. Tests used in one species may not be extrapolated to another (Shimizu et al., 2003). While research has previously described normal cycling pregnancy monitoring in the Bornean orangutan using urinary hormones (Aramaki et al., 2010), no specific guidelines on hormonal cycle tracking and appropriate mating periods have been published so far. In this research, E1g in urinary samples from two Bornean orangutans housed at the Taipei Zoo were analyzed, determining the optimal mating timing for a successful pregnancy. Urinary P4 levels were analyzed for pregnancy monitoring parturition determination. A mating guideline was proposed following our findings, which would take animal welfare into consideration and assist in conservation of the species.
Sample collection
Two female Bornean orangutans were enrolled in this study. One (Xiangniu) was 18 years old and the other (Kexiu) was 21 years old at the time of the study. Both orangutans were kept in sight and smell of male orangutans throughout the research, although they were only housed with males during estrus. Their urine samples were collected at 9:00 every morning to decrease variation due to daily cycling (Hodges Heistermann, 2011). Each sample was aliquoted into two parts. One was sent to Taipei Union Clinical Laboratory for creatinine measurement, while the other was kept for in-house analysis of E1g and P4 by enzyme immunoassay. E1g was monitored for four cycles for both animals. P4 was monitored for the entire course of Xiangniu’s pregnancy.
Hormone analysis
The methodology was graphically presented in Fig 1 and the material details were listed in Supplementary table 1. The standard references were carried out with ten two-fold serial dilutions of an E1g standard beginning from 10 ng/ml and a P4 standard beginning from 4 ng/ml. Creatinine was used to normalize the data in order to minimize the effects of varying hydration levels. All detected hormone levels were divided over creatinine levels and presented as ng of hormone per mg of creatinine.

Fig 1: A graphical representation of the methodology for E1g measurement. For P4 measurement, agoat anit-mouse antibody, bP4 standard and cP4 antibody were used instead. The origins of the materials the equipment were listed in Supplement 1.

Supplementary Table 1: Origins of the materials the equipment.

Data analysis
Data points that had extremely low levels of creatinine were considered to have been contaminated by drinking water and were voided. The obtained E1g levels were plotted against time. Based on our observation, the E1g peak should satisfy two conditions to determine an estrus. Firstly, the data from three days prior to the peak must be at least 1.5 times above the baseline. Secondly, the peak value must be at least twofold over the average of the previous seven days. Peaks that did not fulfill both conditions were excluded in order to prevent misinterpretation of random errors as false peaks. Data points within 14 days before and after the peak were normalized by dividing them with the value of their respective peaks. For the P4 values, a rolling average over previous three data points was used to mitigate random variation. 
E1g cycling
The peaks were pegged as day 0 and data from 14 days before and after the peak were designated as -14 to -1 and 1 to 14, as shown in other studies (Munro et al., 1991). The normalized E1g cycling plots of both animals were merged together in a single graph (Fig 2). The general trend observed was similar with other studies (Inaba et al., 1983; Shimizu, 2005). There is a gradual increase starting from -3 days and a sharp peak is observed at day 0. The average values after day 0 are slightly higher than days before day 0. This is consistent with increased levels of estrogen during the luteal phase (Noakes et al., 2009).

Fig 2: Median values of urinary E1g levels in two Bornean orangutans, normalized against the peak (day 0). The error bar shows quartiles. Days -3 to -1 have values that are 1.5 times over the baseline. Values after the peak (1 to 14) have a higher average than values before the peak (-14 to -1).

P4 pregnancy tracking
Xiangniu was successful in achieving pregnancy. Her P4 levels are presented in Fig 3. The P4 baseline for this individual was below 10 ng/mg creatinine. In the normal non-pregnant cycles, progesterone levels would rise slightly after the estrous peak and then returned to the baseline within 14 days. In pregnancy, the P4 levels did not drop to baseline. Instead, it steadily grew to values above 30 ng/mg creatinine, which were unprecedented for Xiangniu while not pregnant. Throughout the gestation, P4 slowly rose, peaking at values of between 600-700 ng/mg creatinine before dropping. Parturition was achieved 12 days after the drop. P4 values reached baseline values around a week after parturition. A trace of the whole pregnancy is shown in Fig 3.

Fig 3: Rolling average of urinary P4 levels after a successful mating in a Bornean orangutan. Mating occurred on 19/2/2017. Urinary P4 levels gradually increased above baseline and rose to peaks of 600-700 ng/mg creatinine. P4 values started dropping after 8/10/2017 and successful parturition was achieved on 21/10/2017.

General findings
Between the two animals, Xiangniu had consistently higher E1g levels than Kexiu. However, when matched peak-to-peak normalized, similar patterns were observed. The data closely matched other animals (Noakes et al., 2009). Due to different metabolism rates, estrus in relation to the urinary estrogen peak will vary between species (Noakes et al., 2009). In this study, consensual mating was observed ranging from 3 days before to 3 days after the peak. Xiangniu’s successful mating was observed 3 days after the peak.

In humans, pregnancy has been shown to occur from intercourse within 6 days before ovulation (Wilcox et al., 1995). In the rhesus monkey (Macaca mulatta), optimal mating was observed to be within a day of serum estrogen peak, whereas in bonnet monkeys (Macaca radiata), optimal mating was observed to be within a day before after serum estrogen peaks (Parkin Hendrickx, 1975). In previous studies on the orangutan, successful mating was carried out 3 days before urinary estradiol-17β peak (Aramaki et al., 2010). Our mating guideline recommended mating between -3 days to 3 days after the urinary E1g peak. An increase of 1.5 times above the baseline on the -3 day could be an early indicator of upcoming E1g peak and ready to be mated. After mating, progesterone levels can be used for pregnancy monitoring. Pregnancy can be confirmed if progesterone levels continue to rise after 14 days post-mating. Parturition can be expected within two weeks once progesterone levels start to decline. The proposed mating guideline is illustrated in Fig 4.

Fig 4: A flow chart of recommended mating guidelines for the Bornean orangutan.

The antibody used in this study was previously used to analyze hormone levels of the giant panda (Ailuropoda melanoleuca). It was shown here that these antibodies can be also used to accurately track sex hormone levels in the Bornean orangutan.

Due to the small number of animals in this study, further studies with a larger number would be recommended. Other factors that may need to be studied may include different climates or diets, which may also influence the cyclicity of the sex hormones.

  1. Ancrenaz, M., Gumal, M., Marshall, A.J., Meijaard, E., Wich, S.A. Husson, S. (2016). Pongo pygmaeus. The IUCN Red List of Threatened Species. Retrieved from

  2. Aramaki, Y., Hama, N., Kawakami, H., Shimada, Y., Nakane, N., Nakatani, R., Takeda, M., Sano, Y., Hisada, H. Kusunoki, H. (2010). Two successful breeding events timed by monitoring urinary steroid hormones in captive Bornean orangutan (Pongo pygmaeus). Japanese Journal of Zoo Wildlife Medicine. 15(2): 49-55. 

  3. Atmoko, S.U., Setia, T.M., Goossens, B., James, S., Knott, C., Morrogh-Bernard, H., VanSchaik, C. vanNoordwijk, M. (2009). Orangutan mating behavior strategies. In Orangutans: Geographic Variation in Behavioral Ecology Conservation. Oxford University Press. pp. 235-244.

  4. Habumuremyi, S., Robbins, M.M., Fawcett, K.A. Deschner, T. (2014). Monitoring ovarian cycle activity via progestagens in urine feces of female mountain gorillas: a comparison of EIA LC–MS measurements. American Journal of Primatology. 76(2): 180-191. 

  5. Hodges, J.K. Heistermann, M. (2011). Field endocrinology: monitoring hormoral changes infree-ranging primates. In Field Laboratory Methods in Primatology: A Practical Guide. Cambridge University Press. pp. 353-366.

  6. Inaba, T., Imori, T. Saburi, T. (1983). Urinary estrogen levels during the menstrual cycle of the orangutan. Japanese Journal of Veterinary Science. 45(6): 857-859. doi:10.1292/jvms 1939.45.857

  7. Kinoshita, K., Kuze, N., Kobayashi, T., Miyakawa, E., Narita, H., Inoue-Murayama, M., Idani, G. Tsenkova, R. (2016). Detection of urinary estrogen conjugates creatinine using near infrared spectroscopy in Bornean orangutans (Pongo pygmaeus). Primates. 57(1): 51-59. 

  8. Knott, C. D. (1996). Field collection preservation of urine in orangutans chimpanzees. Tropical Biodiversity. 4(1): 95-102.

  9. Munro, C. J., Stabenfeldt, G., Cragun, J., Addiego, L., Overstreet, J. Lasley, B. (1991). Relationship of serum estradiol progesterone concentrations to the excretion profiles of their major urinary metabolites as measured by enzyme immunoassay radioimmunoassay. Clinical Chemistry. 37(6): 838-844. 

  10. Nadler, R. D. (1995). Sexual Behavior of Orangutans (Pongo pygmaeus). In:, The Neglected Ape. [Nadler, R.D., Galdikas, B.F.M., Sheeran, L.K. Rosen N. (Eds.)]. Boston, MA: Springer US. pp. 223-237.

  11. Noakes, D. E., Parkinson, T.J. England, G.C.W. (2009). Veterinary Reproduction Obstetrics. United Kindom: Saunders Elsevier.

  12. Parkin, R.F. Hendrickx, A.G. (1975). The temporal relationship between the preovulatory estrogen peak the optimal mating period in rhesus bonnet monkeys. Biology of Reproduction. 13(5): 610-616. 

  13. Shimizu, K. (2005). Studies on reproductive endocrinology in non-human primates: application of non-invasive methods. Journal of Reproduction Development. 51(1): 1-13. 

  14. Shimizu, K., Udono, T., Tanaka, C., Narushima, E., Yoshihara, M., Takeda, M., Tanahashi, A., et al (2003). Comparative study of urinary reproductive hormones in great apes. Primates. 44(2): 183-190. 

  15. Wilcox, A.J., Weinberg, C.R. Baird, D.D. (1995). Timing of sexual intercourse in relation to ovulation-effects on the probability of conception, survival of the pregnancy, sex of the baby. New England Journal of Medicine. 333(23): 1517-1521. 

Editorial Board

View all (0)