Estradiol is known to have a variety of biological and behavioral effects, but monitoring its function is complex given the many factors influencing its variation. This necessitates large sample sizes which are challenging in captive and wild situations. This study validates the use of opportunistically collected urinary estradiol levels (E2) for use in reproductive monitoring and behavioral research in bonobos (Pan paniscus). We analyzed frozen urine samples from four Columbus Zoo adult females over four years for estradiol and creatinine concentrations (n=117). While E2 was significantly higher in pregnant vs. non-pregnant females (F=66.30, df= 1, p<0.001) it was not significantly different between lactating and regularly cycling females (F=0.40, df= 1, p=0.5304). Among the regularly cycling females, there was a significant positive regression between E2 and sexual swelling size (F= 4.43, df=1,81, p=0.0384). No differences in E2 variation were detected between individuals in this study. Specifically, when the amount of variation in estradiol due to sexual swelling was statistically controlled for, there was no significant effect of age (n=83, r=0.08059, p=0.4689) or rank (n=83, r=0.1361, p=0.22) on estradiol variation. Overall, these findings indicate that opportunistically sampled urinary estradiol can be paired with visual observation to help detect changes in reproductive status. The shift from lactational amenorrhea back to estrogen cycling may be less clearly defined than expected, and uneven sampling may exacerbate difficulty in detecting some of the more subtle shifts in estradiol levels. While it is known that extended maximal tumescence in bonobos may function to obscure the exact date of ovulation, we did confirm that ratings of visual tumescence still provide useful information regarding relative estradiol levels. By publishing more methodologies and results of this kind, we hope to promote the continued study of estradiol in bonobos as it is relevant to both health monitoring and behavioral research goals.

Subjects for this study included four adult female bonobos housed at the Columbus Zoo and Aquarium (CZA) in Columbus, Ohio, USA. Samples were collected for these individuals during the summer months of 2012, 2013, 2014, and 2015. Urine samples were collected by CZA staff either by using a free catch method in which individual subjects urinated through the mesh caging directly into a collection cup, or, by pipetting urine directly off a clean floor surface into a plastic cryo-tube. All subjects had received previous training to urinate on command in exchange for a small food reward. 

Upon collection, samples were immediately frozen and stored at -20°C until they were packed on dry ice and shipped overnight to the Global Health Biomarker Lab at the University of Oregon in Eugene, OR where they were stored at -80°C until time of analysis. 

For analysis, samples were brought to room temperature and diluted (1:4) in assay buffer supplied in the 96-well Detect X® Estradiol ELISA Kit from Arbor Assays™ (catalog no. K030-H) and assayed according to the kit manufacturer’s instructions. The manufacturer of this kit reported the detection limit for this assay as 39.6 pg/mL. This Arbor Assays ELISA protocol is validated for use with urinary samples as well as with human and animal models. Each plate allows for 40 samples to be run in duplicate.

To control for variation in the amount and concentration of urine samples, all samples were assayed for creatinine concentrations using DetectX® Urinary Creatinine Detection Kits from Arbor Assays™ (catalog no. K002-H). The manufacturer listed the detection limit for this assay as 0.019 mg/dL. Samples run for creatinine were diluted (1:20) and run according to the kit manufacturer’s instructions. All plates were read using a BioTek™ microplate reader and analyzed with Gen5™ software version 2.0.

Of the frozen urinary samples available, we were able to assay a total of 31 samples from Ana Neema, 16 from Lady, 48 from Susie, and 22 from Unga for a total of 117 samples. The estradiol concentration values obtained from these assays were corrected for creatinine concentration by dividing the estradiol concentration value of each sample by its respective creatinine value.