Behav Ecol Sociobiol DOI 10.

1007/s00265-012-1345-0

ORIGINAL PAPER

Giant pandas attend to androgen-related variation in male bleats

Benjamin D. Charlton & Ronald R. Swaisgood & Zhang Zhihe & Rebecca J. Snyder

Received: 15 January 2012 / Revised: 1 March 2012 / Accepted: 5 March 2012 # Springer-Verlag 2012

Abstract Although androgen-dependant traits are predicted to signal overall male quality, no study has examined the response of a nonhuman animal to variation in a known acoustic cue to male androgen levels (steroid hormones that are key drivers of male sexual behaviour). Here, we use a single-speaker approach to present male and female giant pandas with re-synthesised male bleats representing callers with high and low androgen levels. Our results revealed that male and female giant pandas had significantly greaterlooking responses, spent more time pacing, and were faster to respond to playbacks of bleats simulating high androgen males. When we analysed the sexes separately, a slightly different response pattern was revealed: whereas males and females still had significantly greater-looking responses and were faster to respond to bleats simulating high androgen males, only male giant pandas tended to spend more time pacing. These findings suggest that vocal cues to male androgen levels are functionally relevant to male and female
Communicated by E. Huchard B. D. Charlton : R. J. Snyder Zoo Atlanta, Atlanta, GA, USA R. R. Swaisgood Applied Animal Ecology, San Diego Zoo Institute for Conservation Research, San Diego, CA 92027-7000, USA Z. Zhihe Chengdu Research Base of Giant Panda Breeding, Chengdu, Sichuan Province, Peoples Republic of China Present Address: B. D. Charlton (*) Department of Cognitive Biology, University of Vienna, Vienna, Austria e-mail: benjamin.charlton@univie.ac.at

giant pandas during the breeding season, and constitute the first demonstration that a nonhuman animal could be using a vocal signal to assess male hormonal state. We go on to discuss the ecological relevance of signalling androgen levels in this species sexual communication and the possible application of our results to conservation breeding. Keywords Giant pandas . Acoustic cues . Androgens . Vocal communication

Introduction Androgen-dependant traits are predicted to signal overall male quality (Folstad and Karter 1992; Zahavi and Zahavi 1997) and female mate choice based on them is well documented (Andersson 1994). Among mammals, darker male coloration indicates higher androgen levels in rhesus macaques (Macaca mulatta) and African lions (Panthera leo; Rhodes et al. 1997; West and Packer 2002), and in both these species females prefer darker male variants (West and Packer 2002; Waitt et al. 2003). These findings are consistent with the hypothesis that these visual traits reliably signal male quality; however, mammal vocal signals may also provide reliable information to receivers about the callers hormonal quality in reproductive contexts. For example, in humans, low voice pitch is predictive of high testosterone and females find lower-pitched voices more attractive (Collins 2000; Feinberg et al. 2005; Puts 2005). In addition, males consider lowpitched voices to be more dominant (Puts et al. 2007) and, presumably, more threatening. Nonhuman animals might use vocal cues to male hormonal quality to make similar judgements for intrasexual assessment and mate choice (Fusani et al. 1994; Galeotti et al. 1997; Koren and Geffen 2009; Pasch et al. 2011). Despite this, however, no study has examined the

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response of any nonhuman animal to systematic variation in a known acoustic cue to male androgen levels, i.e. while holding all other acoustic parameters constant. Giant pandas (Ailuropoda melanoleuca) are solitary mammals that rely on effective communication to coordinate their annual breeding activities (Schaller et al. 1985). Previous studies have demonstrated the importance of olfaction in this species sexual communication and indicate that chemical signals could be used to recruit mating partners (Swaisgood et al. 1999, 2002), stimulate male sexual motivation (Swaisgood et al. 2000), and assess potential rivals (White et al. 2002, 2003). However, giant pandas are also highly vocal during the breeding season (Schaller et al. 1985). In particular, male giant pandas display their highest rates of bleating during encounters with oestrous females (Peters 1982; Kleiman 1983), suggesting that this vocalisation is crucial for coordinating mating activity. Recent work has shown that male bleats contain information on the callers identity and body size (Charlton et al. 2009c, b), and that male and female giant pandas attend to this information (Charlton et al. 2009a, 2010b). In addition, the rate of fundamental frequency (F0) modulation in male giant panda bleats is positively correlated to male androgen levels (Charlton et al. 2011); raising the possibility that male and female giant pandas could also make use of this information in reproductive contexts. Charlton et al. (2011) suggest that higher androgen levels could increase vocal fold stiffness, by increasing the collagen/elastin ratio (Fischer and Swain 1980), leading to greater elastic recoil and higher potential rates of F0 modulation. Furthermore, given that males with higher levels of testosterone (the primary androgen secreted by the testis) are typically more aggressive (Zielinski and Vandenbergh 1993; Gerald 2001) and preferred by females in mate choice contexts (for a review, see Clutton-Brock and McAuliffe 2009), acoustic features that signal male androgen levels may have utility in giant panda sexual communication for both intrasexual assessment and mate choice. Here, we examined the response of male and female giant pandas during the breeding season to androgen-related variation in male bleats. Specifically, we played back male giant panda bleats that had been re-synthesised to have high and low F0 modulation rates, simulating male callers with high and low androgen levels, respectively (Charlton et al. 2011). High testosterone levels are associated with increased sperm quality (Minter and DeLiberto 2008), and may signal overall male quality because androgens are immunosuppressant and can only be produced at high levels by otherwise healthy individuals (Folstad and Karter 1992; Zahavi and Zahavi 1997). Consequently, we predicted that giant pandas would respond more strongly to bleats simulating males with high androgen levels, representing high-quality mates and more competitive/dangerous rivals. Our findings will raise further questions about the adaptive function of androgen signalling

in this highly endangered species sexual communication, and may have application to conservation breeding.

Methods Experimental site and animals The playback experiments were conducted at Chengdu Research Base of Giant Panda Breeding (CRBGPB), Chengdu, Peoples Republic of China, during February to May 2009. A total of 14 adult giant pandas (eight females and six males) were used as subjects in the playback experiments. The subjects were fed a diet of bamboo, panda cakes (containing nutritional supplements) and apples twice daily, and housed individually in enclosures with an indoor bedroom measuring approximately 53 m and an outdoor yard measuring approximately 20 m2. The playback experiments were carried out when the subjects were in their respective outdoor yards. Playback stimuli To create the playback stimuli, we selected 10 single giant panda bleats from each of four adult males that were aged 1017 years (mean013.5 years) and unfamiliar to the current residents at the CRBGPB. These males had been previously recorded at four different institutions (CRBGPB, China, Zoo Atlanta, San Diego Zoo, and Smithsonians National Zoological Park, USA) using an Audio-Technica AT835b microphone and a TASCAM HDP2 portable solidstate digital recorder at distances ranging from 3 to 10 m. The recordings were transferred to an Apple Macintosh Macbook computer, saved as AIFF files and normalised to 100% peak amplitude (sampling rate: 48 kHz, amplitude resolution: 16 bits). The playback sequences consisted of 10 single bleats from a given male exemplar each separated by 4 s (mean total duration044.8 s; range043.747.4 s). Male giant pandas often give long series of bleats when they interact with conspecifics during the breeding season, and 4-s gaps between these calls constitutes a realistic rate of delivery (B. D. Charlton, personal observation). The mean intensity of all the playback sequences was scaled to 70 dB. Acoustic analyses To determine the appropriate re-adjustment factors by which to re-synthesise the bleat stimuli, we initially measured the F0 modulation rate for each bleat using Praat 5.0.29 DSP package (http://www.praat.org/). The mean F0 values for each bleat were extracted using a forward cross-correlation (To pitch (cc) command) algorithm in Praat. The time step in the analysis was 0.01 s, and to limit the possibility of octave jumps, the minimum and maximum values for F0

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Re-synthesis of playback stimuli In a previous study, the minimum and maximum values for male bleat F0 modulation rate were 7.36 and 11.4 modulations per second, respectively (Charlton et al. 2009c). Therefore, in order to realistically simulate natural variation in F0 modulation rate we created slow and fast F0 modulation variants with rates of eight and 11 modulations per second. Since the rate of F0 modulation in male giant panda bleats is positively correlated to male androgen levels (Charlton et al. 2011), these values correspond to male giant pandas with low and high androgen levels, respectively. The re-synthesis factors for these target values were deduced by dividing the intended value by the original measured value for each bleat. We then used a pitch synchronous overlap add method-based algorithm embedded in Praat to adjust the duration of our experimental stimuli by these factors. This effectively changed the F0 modulation rates by the required amount without changing any other spectral features. Finally, so that the duration of both F0 modulation rate variants for a given bleat were the same, we cut the longer of the two files to equal the duration of the shorter F0 modulation rate variant and used the fade out facility in Praat to ensure the stimuli sounded realistic (see Fig. 1). This gave us eight unique playback sequences, representing the two different male androgen variants for each of the four male exemplars. Playback experiments Playbacks were initiated when subjects were settled and their attention was directed away from the speaker position. The eight re-synthesised bleat playback sequences were presented using an Anchor Audio Explorer Pro 6000 loudspeaker at sound pressure levels sounding equivalent to that of naturally bleating males of 75 dB SPL at 1 m from the source (determined using a Radio Shack Sound Level Meter, set for C-weighted fast response). Each subject received a total of two playbacks, representing the two playback conditions (low and high androgen male) from the same male exemplar. To minimise habituation, playbacks to a given individual were separated by 13 days (mean0 1.2 days). The presentation order was alternated across the experiment, and the use of a given male exemplar was balanced across subjects, so that each exemplar was used three or four times in the experiment (two exemplars were
0 0 5000 F0

Frequency (Hz)

were set according to the F0 contour as observed on the spectrogram. A five-point average smoothing filter was used to remove any rapid variations caused by analysis imprecision before the number of complete cycles of F0 modulation across a bleat was measured. We then obtained the F0 modulation rate per second by dividing the number of F0 modulations in a bleat by the calls duration in seconds.

5000

Time (s)

0.72

Frequency (Hz)

0 0

F0

Time (s)

0.72

Fig. 1 Spectrograms of re-synthesised bleat stimuli from one of the four male exemplars used in the playback experiments (spectrogram settings, FFT method; window length, 0.03 s; time steps, 250; frequency steps, 1000; Gaussian window shape; dynamic range, 50 dB). The upper panel shows the slow F0 modulation variant, simulating a male with low androgen levels. The lower panel shows the same bleat that has been re-synthesised to represent a male with high androgen levels. Note that the rate of F0 modulation increases as you go from the low to the high androgen male condition, from eight to 11 modulations per second, respectively. The arrow denotes the position of F0

used four times and two three times). A Sony hard drive digital camera (model DCR-SR42) was used to capture behavioural responses during the playback and for 2 min after each playback presentation (the experimental period). Behavioural and statistical analysis The videotapes were analysed frame-by-frame (frame 0 0.04 s) using SportsCode GameBreaker 7.0.121 digital video analysis system for Mac OS 10 (SportsTec, Sydney). In order to quantify the response of male and female giant pandas to each playback condition, we measured the total amount of time during the experimental period that each subject spent looking towards the speaker position whilst stationary (looking), the amount of time spent pacing (pacing), and the time between the onset of the playback and the first response (latency). Because it was not possible to normalise the data distribution for all the response variables,

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Wilcoxon signed-ranks tests were used to determine if significant differences existed between the two experimental conditions (low and high androgen male variants). Due to the low sample sizes, we calculated exact P values (Mundry and Fischer 1998). In addition, we examined the responses of male and female giant pandas separately in order to reveal any sex-related differences in their direction of response to our two classes of playback stimuli. Two subjects that failed to look in response to the playback were assigned latency values of 170 s. This value just exceeded the maximum time given to respond after playback initiation (longest playback sequence047.4 s+120 s observation period). IBM SPSS statistics version 19.0.0 for Mac OS X was used to run the statistical tests, significance values were set at 0.05 and twotailed probability values are quoted.

Results Giant pandas spent significantly more time looking towards the speaker and pacing when presented with bleats simulating males with high androgen levels, than when they were presented with bleats simulating low androgen males (see Table 1). In addition, subjects were significantly faster to respond when presented with the high androgen male condition (see Table 1). When we analysed the sexes separately, both males and females still had significantly greater-looking responses and were faster to respond to bleats simulating high androgen males (see Table 1), and a trend for male giant pandas to spend more time pacing in response to this class of playback stimuli was revealed (P00.063; see Table 1).

Discussion We have shown that male and female giant pandas respond to androgen-related acoustic variation in male bleats. Interestingly, whereas both male and female giant pandas had greater-looking responses and were faster to respond to bleats simulating high
Table 1 Paired comparisons of behavioural responses to the two playback conditions: high versus low androgen male variant

androgen males, only males tended to spend more time pacing. Our re-synthesis techniques and repeated measures design (in which each subject received the same exemplar) ensure that these differential responses could only be due to the shift in F0 modulation rate between the experimental conditions, representing either a high or low androgen male (see Fig. 1). While it is important to note that the F0 modulation rate of male bleats could be affected by other factors, these findings suggest that male and female giant pandas use these vocalisations to assess male androgen levels and hence, that vocal cues to male hormonal state are functionally relevant to giant pandas during the breeding season. Although it is difficult to unequivocally establish the significance of looking and latency responses, the greaterlooking responses and lower response latencies of female giant pandas to male bleats simulating callers with high androgen levels may indicate a sexual preference (Waitt et al. 2003). Androgens are reported to have energetic costs (Wingfield et al. 1990) and immunosuppressant effects (Folstad and Karter 1992) and, therefore, only males with high energy reserves and natural disease resistance can endure the costs associated with having high levels (Zahavi and Zahavi 1997). Studies of wild giant pandas indicate that males of this energy-limited species may not be able to sustain elevated testosterone levels and, in contrast with most polygynous seasonally breeding mammals, show only brief pulses of elevated testosterone in response to the social challenge of courtship and intra-sexual competition (Nie et al. 2011). Testosterone may be more costly for male pandas than other species, and may therefore be a more sensitive indicator of male quality. In addition, androgen levels are positively correlated with sperm quality in other seasonally breeding mammals (Hellgren et al. 1989; Brown et al. 1991; Monfort et al. 1993; Minter and DeLiberto 2008). Consequently, females that are mated by males with high androgen levels might gain indirect benefits by providing offspring with increased heritable resistance to pathogens, heritable qualities leading to increased foraging or energetic efficiency, and even the direct benefits of increased likelihood of conception. It is also worth

Response (s)

High androgen variant (meanSE) 21.145.49 19.117.19 5.881.98 21.137.02 28.6112.30 8.114.13 21.158.45 11.998.38 4.20.65

Low androgen variant (meanSE) 7.142.31 1.981.12 50.3715.86 8.323.66 2.411.57 39.8818.18 6.253.15 1.661.66 58.2324.89

Z 3.233 2.240 2.982 2.201 2.023 2.201 2.380 1.069 2.240

Exact P values are used

Looking Pacing Latency Male looking Male pacing Male latency Female looking Female pacing Female latency

<0.001 0.023 0.001 0.031 0.063 0.031 0.016 0.500 0.023

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noting that dominant male giant pandas (perhaps larger individuals and/or those with higher testosterone levels) may monopolise access to females during the breeding season or at peak conception time (Soltis 1999; Preston et al. 2003), and this could limit any female choice behaviour that might otherwise occur (as suggested by Charlton et al. 2010a). The greater-looking responses, lower response latencies, and tendency to spend more time pacing displayed by male giant pandas in response to bleats representing high androgen males may reflect their motivation to avoid, or even to locate rivals with high androgen levels that are likely to represent a greater threat to them (Zielinski and Vandenbergh 1993). Indeed, studies on this species olfactory communication have shown that giant pandas are able to assess a signallers competitive ability from scent deposits and appear to avoid males of higher status (White et al. 2002), strongly suggesting that this type of information is important to them in reproductive contexts. Furthermore, male giant pandas may be more likely to approach bleats simulating high androgen males if they themselves have high circulating androgen levels and hence, are less threatened by these individuals. Although we do not have the data to support this contention, future giant panda studies could investigate how male responses to androgenrelated variation in male bleats vary according to their own intrinsic hormonal state. An additional next step is the potential application of our results to conservation breeding. Insufficient or inappropriate opportunities for communication are often a contributing cause to reproductive failure in conservation breeding programmes (Swaisgood and Schulte 2010) and critical knowledge of cues to mate quality available in giant panda vocalisations, coupled with an understanding of how giant pandas respond to variation in them, suggests a way forward. For example, some mating failures might be attributed to females choosing not to mate with males whose vocal signals indicate low androgen levels. As a result, it may be possible to enhance sexual motivation and/or guide mate choice decisions by presenting females with re-synthesised bleats representing high androgen males, encouraging them to mate with individuals that are deemed appropriate by conservation managers. Experimental manipulation of mate choice has been conducted successfully on other mammals using chemical signals (Fisher et al. 2003; Roberts and Gosling 2004) and by supplementing natural communication in this way, conservation managers could include under-represented males in the breeding programme and thus, facilitate the goals of genetic management. In conclusion, research on giant panda olfactory communication has provided meaningful insights into this species reproductive behaviour (for an overview, see Swaisgood et al. 2004) but the potential role of vocal communication remains under-investigated. Our findings emphasise the importance of male giant panda bleats in this highly endangered species

sexual communication and point the way for further research. We suggest that future studies use a playback approach to show how vocal cues in giant panda bleats influence mate choice decisions, but also how acoustic, olfactory and other modalities act in concert to coordinate the reproductive process in giant pandas. Conservation science for giant pandas has come a long way in recent years (Swaisgood et al. 2010) but we must continue to advance biological knowledge to address the many conservation problems facing this iconic species. By identifying key aspects of reproductive behaviour in giant pandas, we can hope to provide the ideal environments and stimuli required for them to reproduce, and in doing so increase the success of captive breeding programmes.
Acknowledgments We thank the keepers and staff at the CRBGPB. This material is based upon work supported in part by the STC Program of the National Science Foundation under Agreement No. IBN-9876754. Ethical Standards This work follows the Association for the study of Animal Behaviour/Animal Behaviour Society guidelines for the use of animals in research. In addition, the procedures used in the research did not affect the housing or management of the animals and comply with the law of the Peoples Republic of China.

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