The novel use of urinary androgens to optimise detection of the fertile window in giant pandas

Abstract

Abstract: Giant pandas are mono-estrus seasonal breeders, with the breeding season typically occurring in the spring. Successful fertilization is followed by an embryonic diapause, of variable length, with birth in the late summer/autumn. There is a need for additional understanding of giant panda reproductive physiology, and the development of enhanced biomarkers for impending proestrus and peak fertility. We aimed to determine the utility of non-invasive androgen measurements in the detection of both proestrus and estrus. Urine from 20 cycles (-40 days to +10 days from peak estrus) from 5 female giant pandas was analyzed for estrogen, progestogens and androgens (via testosterone and DHEA assays), and hormone concentrations were corrected against urinary specific gravity. Across proestrus, estrogens increased while progestogens and androgens decreased - at the point of entry into proestrus, androgens (as detected by the testosterone assay) decreased prior to progestogens and gave 4 days advanced warning of proestrus. At the time of peak estrus, androgens (as detected by the DHEA assay) were significantly increased at the time of the decrease in estrogen metabolites from the peak, acting as an alternative confirmatory indicator of the fertile window. This novel finding allows for enlargement of the preparative window for captive breeding and facilitates panda management within breeding programmes. Androgens allow an enhanced monitoring of giant panda estrus, not only advancing the warning of impending proestrus, but also prospectively identifying peak fertility.

Lay summary: Giant pandas have one chance at pregnancy per year. The 2-day fertile window timing varies by year and panda. This is monitored by measuring the level of estrogens in the urine, which increase, indicating an upcoming fertile period. After 1-2 weeks of increase, estrogens peak and fall, marking the optimal fertile time. We tested other hormones to see if we can predict the fertile window in advance, and the specific fertile time with more accuracy. In 20 breeding seasons from 5 females, we found androgens, usually thought of as male hormones, had an important role. Testosterone gives 4 days advanced warning of estrogens increasing. DHEA identified peak estrogen and the fertile time before needing to see a confirmed decrease in estrogen itself. Therefore, androgens help improve monitoring of the giant panda breeding season, giving early warning of fertility, key in facilitating captive breeding and giant panda conservation.

Keywords: ELISA; breeding; estrus; female; hormones; non-invasive.

Figure 1

Parallelism assessment of giant panda urine (n = 3) in comparison with the testosterone (A) and DHEA (B) standard curves. A good parallelism was achieved for giant panda urine with dilutions between × 2 and × 32. Freeze-thaw cycle assessment of giant panda urine (n = 3) for both neat urine and a × 5-pre-diluted urine as tested for both testosterone (C) and DHEA (D) following up to six freeze-thaw cycles. There was no significant difference for either neat or pre-diluted urine at any number of freeze-thaw cycles in comparison to the original hormone concentrations of the samples (testosterone P = 0.70; DHEA P = 0.91). Monthly average testosterone and DHEA (E) profiles ± s.e.m. for a castrated male giant panda from the 22 months prior to castration until 13 months following castration. The black arrow indicates the month of tumour discovery, the red arrow indicates the month of castration. The November 2018 monthly average is indicative of the intact male, the first urine samples from post-castration were from December 2018.

Figure 2

The combined average hormone profiles of estrogens (E), progestogens (P) and androgens (testosterone (T) and DHEA) ± s.e.m. over the period of interest aligned to the day of peak estrogen. Note the split y-axis scale on the E/P/T axis to allow for clear visualization of the combined profiles at the timing of the entry into proestrus, and the separate y-axis for DHEA. The grey line at day -11 indicates the average start of the proestrus period based on when the concentration of estrogen increased 2 s.d. above the mean concentration. The E/P crossover is highlighted in red, occurring between day -10 and -9 following the aforementioned entry into proestrus. The E/T crossover is highlighted in yellow, occurring between day -14 and -13. The day of peak estrus (day 0) is highlighted in blue.