Electronic supplemental methods - Proceedings of the Royal Society B

Definition of female fertile phase based on endocrine data
Regular urine samples were collected on most days and several times a day as
females approached peak oestrus. Urine samples were aspirated from enclosure
floors and then indexed by creatinine to adjust for variability in the urine dilution,
before being frozen at -20 degrees C until analysis at the endocrinology
laboratory of the Wolong research base. When multiple samples were taken the
average oestrogen values for each day were used for the analysis. Assays of
estrone-3-glucuronide were measured using enzyme-linked immunosorbent
assay (EIA) as previously described (Czekala et al. 2003; McGeehan et al.
2002). This method uses a solid phase assay with antibody-coated microtiter
plate wells (antisera made against estrone-3-glucuronide: 100 ul, 1:5000 UCDavis, Davis, CA) used in competition with estrone 3-glucuronide HRP (UCDavis, Davis, CA). The colimetric assay was finalised with the addition of the
substrate tetramethylbenzidine (Sigma, St Louis, MO).
To account for an
excretion lag time of approximately 12-24 hours oestrogen levels were backdated
one day. This method has been used in previous studies on giant pandas
(Czekala et al. 2003; McGeehan et al. 2002; Lindburg et al. 2001). Moreover,
although the lag time for the excretion of urinary oestrogen metabolites in this
species has not been established, peak faecal glucocorticoid concentrations are
detected approximately 12 hours after peak serum levels (Kersey 2009) and
other mammals typically excreted urine steroids on the same day as the peak in
serum (Wasser et al. 1994), justifying the use of the urinary peak in this analysis
as denoting a significant change in ovarian activity.
In giant pandas, a greater than six-fold increase in urinary oestrogen above
basal, followed by a rapid decline to baseline concentrations is indicative of
ovulation (Steinman et al. 2006) (female oestrogen profiles are provided as
electronic supplemental material). Using these criteria we could determine the
date of ovulation (designated Day 0 of the reproductive cycle) and group
recordings relative to this. In humans, conception probably peaks just before
ovulation and the median ovum survival time is approximately 12 hours (Lynch et
al. 2006). However, sperm can be fertile for up to 72 hours (Wilcox et al. 1995)
and hence, any copulations occurring up to three days before ovulation can still
be fertile. Based on this, we defined the ‘fertile’ phase of the female oestrous
cycle as the three days prior to ovulation and the date of ovulation itself (-3 to 0
days) and the ‘pre-fertile’ phase as the six days (-4 to -9) immediately preceding
this; giving us 219 ‘pre-fertile’ and 460 ‘fertile’ chirps for the comparisons.
Czekala, N. M., McGeehan, L., Steinman, K. J., Xuebing, L. & Gual-Sil, F. 2003
Endocrine monitoring and its application to the management of the giant
panda. Zoo Biol. 22, 389-400.
Lindburg, D. G., Czekala, N. M. & Swaisgood, R. R. 2001 Hormonal and
behavioral relationships during estrus in the giant panda. Zoo Biol. 20,
Lynch, C. D., Jackson, L. W. & Buck Louis, G. M. 2006 Estimation of the dayspecific probabilities of conception: current state of the knowledge and the
relevance for epidemiological research. Paediatr. Perinat. Epidemiol.
McGeehan, L., Li, X., Jackintell, L., Czekala, N. M., Huang, S. & Wang, A. 2002a
Evaluating reproductive performance in giant pandas (Ailuropoda
melanoleuca) with behaviour and hormones. Zoo Biol. 21, 449-466.
Steinman, K. J., Monfort, S. L., McGeehan, L., Kersey, D. C., Gual-Sil, F.,
Snyder, R. J., Wang, P., Nakao, T. & Czekala, N. M. 2006 Endocrinology
of the giant panda and application of hormone technology to species
management. In Giant pandas: veterinary medicine and management (ed.
D. E. Wildt, Zhang, A., Zhang, H., Janssen, D. J., Ellis, S.), pp. 198-230.
New York: Cambridge University Press.
Wasser, S. K., Monfort, S. L., Southers, J. & Wildt, D. E. 1994 Excretion rates
and metabolites of estrodiol and progesterone in baboon (Papio
cynocephalus cynocephalus) faeces. J. Reprod. Fertil. 101, 213-220.
Table 1: Rotated component matrix to show how our five response measures
loaded onto each of the two principal components.
The two principal
components explained 86% of the variance; eigenvalues are given in upper part
of the table and the correlations between the response variables and each
principal component in the lower part.
Component 1 Component
Proportion of variance (%)
Cumulative proportion (%)
Response measures
First look duration
Total look duration
Number of looks
Time in proximity zone