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

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.




 
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).


 
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.


 
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.



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.