- Email: [email protected]
Seasonality of fecal androgen and glucocorticoid metabolite excretion in male goral (Naemorhedus griseus) in Thailand
Animal Reproduction Science 146 (2014) 70–78
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Seasonality of fecal androgen and glucocorticoid metabolite excretion in male goral (Naemorhedus griseus) in Thailand Jaruwan Khonmee a,∗ , Janine L. Brown b , Suvichai Rojanasthien a , Dissakul Thumasanukul c , Adisorn Kongphoemphun c , Boripat Siriaroonrat d , Wanlaya Tipkantha d , Veerasak Punyapornwithaya a , Chatchote Thitaram a a b c d
Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Center for Species Survival, Smithsonian Conservation Biology Institute, Front Royal, VA 22630, USA Omkoi Wildlife Sanctuary, Department of National Park, Wildlife and Plant Conservation, Omkoi, Chiang Mai 50310, Thailand Conservation Research and Education Division, Zoological Park Organization, Dusit, Bangkok 10300, Thailand
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 31 January 2014 Accepted 7 February 2014 Available online 19 February 2014
Keywords: Goral Glucocorticoid metabolites Androgen metabolites Non-invasive hormone monitoring Seasonality
a b s t r a c t There is no information on the endocrinology of Chinese goral (Naemorhedus griseus), a high priority species for captive breeding and reintroduction in Thailand. This study characterized fecal androgen and glucocorticoid metabolites in male goral at Omkoi Wildlife Sanctuary to investigate seasonal relationships. Fecal samples were collected 3 days/week for 1 year from eight adult males. Mean androgen metabolite concentrations were greater (P < 0.05) during the rainy season (289.82 ± 9.18 ng/g) and winter (224.09 ± 11.97 ng/g) compared to the summer (195.48 ± 8.23 ng/g), and were related to breeding activity. A similar pattern was observed for glucocorticoid concentrations (22.10 ± 0.72 ng/g compared to 21.98 ± 0.98 ng/g compared to 15.30 ± 0.48 ng/g), respectively, and this resulted in a positive correlation between the two hormones (P < 0.05). There also were positive correlations between fecal androgen metabolite concentrations and temperature (P < 0.05) and day length (P < 0.05). In summary, this is the first study to assess endocrine function in male goral, and results showed seasonal variation in testicular and adrenal steroidogenic function, with greater activity in the rainy season and winter. Given that resources for captive male goral are consistent throughout the year, reproduction may be regulated primarily by photoperiod in this species. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chinese goral (Naemorhedus griseus) are small ungulates with a goat-like appearance that inhabit steep rocky terrain throughout Burma, China, India, Thailand and Vietnam (Rabinowitz and Khaing, 1998; Patton et al., 2000;
∗ Corresponding author. Tel.: +66 53 948 046; fax: +66 53 948 065. E-mail addresses: [email protected], [email protected] (J. Khonmee). http://dx.doi.org/10.1016/j.anireprosci.2014.02.008 0378-4320/© 2014 Elsevier B.V. All rights reserved.
Duckworth et al., 2008). These animals are listed as ‘Vulnerable’ on the IUCN Red List (Duckworth et al., 2008) and are one of 15 protected species under the Wild Animal Reservation and Protection Act of Thailand (Chaiyarat et al., 1999). Goral live in small family groups, with males defending territories of ∼25 ha (Duckworth et al., 2008). In Thailand, this species is found exclusively in protected areas of Northern Thailand, in steep steppe areas and mountainous plateaus (Chaiyarat et al., 1999). The size of wild goral populations is unknown, but numbers are believed to be decreasing due to habitat loss and hunting by local people for meat, fur and
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
traditional medicine (Duckworth et al., 2008). As a result, this is a priority species of the Zoological Parks Organization of Thailand for re-introduction and captive breeding efforts. There are about 100 goral held among three captive facilities in Thailand (Omkoi Wildlife Sanctuary, Chiang Mai Night Safari and Chiang Mai Zoo). Animals are breeding at the first two facilities; however, reproduction is suboptimal and populations are not self-sustaining. There is interest in developing assisted reproductive technologies (ART) for this species, for example artificial insemination and embryo transfer, to supplement captive breeding efforts and facilitate genetic management. Inbreeding is a particular concern as routine transferring of males between facilities for breeding has not been a logistically successful strategy. Zoos and captive breeding are having an increasingly important role in conservation efforts of many threatened species (Conde et al., 2011), but success is predicated on a thorough knowledge of species biology, something that is lacking for goral. Previous studies have focused on observations of sexual behavior, particularly rutting activities in captive male and female Himalayan (Neamorhedus goral; Lovari and Apollonio, 1994) and red (Naemorhedus cranbrooki; Xie, 2006) goral. All indications are that these animals are seasonal, with maximal activity in the winter months. In Chinese goral, breeding has been reported to occur between November and December in the wild (Lekagul and McNeely, 1988) and between October and January at the Choengdoi Suthep Nature and Wildlife Education Centre (Kanbunjong, 1993). However, there are no endocrine data to confirm this seasonality in Chinese or any other goral species. Given the limited amount of reproductive information, and the need to determine optimal times for scheduling ART procedures, studies to characterize androgen and glucocorticoid activity in male goral are warranted. Most studies of wildlife utilize non-invasive steroid metabolite monitoring to assess endocrine function as an alternative to blood sampling (Huber et al., 2003; Millspaugh and Washburn, 2004). Because hormonal metabolites in feces represent an accumulation over time, these metabolites can more accurately reflect average values (Pelletier et al., 2003). Fecal androgen metabolite analyses have been used to characterize seasonal testicular activity in ungulate species (Yamauchi et al., 1997; Li et al., 2001; Pelletier et al., 2003; Mooring et al., 2004).
Table 1 Mean (±SEM) daily temperature, rainfall and day length across seasons at Omkoi Wildlife Sanctuary during the study period. Data were obtained from The Northern Meteorological Center, Meteorological Department, Chiang Mai, Thailand. Season
Average temperature (◦ C)
Rainfall (mm)
Day length (h)
Summera Rainya Wintera
23.47 ± 2.30b 24.59 ± 1.85c 19.77 ± 1.69a
2.92 ± 0.29b 5.19 ± 0.39c 1.40 ± 0.12a
12.28 ± 0.03b 12.76 ± 0.02c 11.20 ± 0.01a
Values differ among seasons, different superscript letters (a,b,c) indicate differences (P < 0.05). a Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
71
Thus, the objectives of the present study were to (1) determine the influence of season on testicular steroidogenic function in Chinese goral by assessing androgen metabolite concentrations in feces, and (2) examine seasonal relationships between fecal androgen and glucocorticoid metabolite excretion and sexual behavior throughout the year. The ultimate goal is to develop appropriate management strategies that can be implemented to propagate a threatened species that is of national importance to Thailand. 2. Materials and methods 2.1. Animals and samples collection All animal procedures were approved by the Animal Ethic Committee Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand (S22/2553). Eight captive-born male goral (average age, 5.29 ± 0.24 years) were housed individually in outdoor enclosures (6 m × 9 m × 3 m) adjacent to females at Omkoi Wildlife Breeding Center in Chiang Mai, Thailand (17◦ 48 4 N, 98◦ 21 31 E). All enclosures had dirt floors, an open shelter and several natural trees for shade. Animals were exposed to natural light, and fed roughage (Panicum grass; Brachiaria mutica), plus concentrate (500 g, Betagro 009 cattle finisher pellet (12% protein, 2% fat, 13% fiber, 13% moisture); Betagro Company Limited, Thailand) and natural leaves (little yellow star, Melampodium divaricatum) once daily with unlimited access to fresh water. Feces were removed every evening, and freshly defecated feces (∼30 g) were collected between 0830 and 0930 h every morning from each male goral 3 days/week for 1 year. Samples were stored at −20 ◦ C until analysis. Enclosures were configured in two rows of eight adjoining pens, alternating between male and female, with a common corridor in between. 2.2. Environmental data There are three defined seasons in Thailand: summer (February 16 to May 15), rainy season (May 16 to October 15) and winter (October 16 to February 15). Information on average temperature (◦ C), amount of rainfall (mm/day) and day length (h) in each month of the study was obtained from The Northern Meteorological Center, Meteorological Department, Ministry of Information and Communication Technology, Chiang Mai, Thailand (Thai Meteorological Department, 2013). There were seasonal differences in average temperature, rainfall and day length at Omkoi Wildlife Sanctuary, being greatest in the rainy season and lowest in the winter (P < 0.05) (Table 1). 2.3. Behavioral data Every morning of the study, each male goral was placed in the common area, which permitted visual and olfactory access to females. Quantitative behavioral data were collected by instantaneous scan sampling during daily 30-min observation sessions. Male sexual behaviors were recorded and included tongue flick, approach to female, smell, lick and flehmen as described for red goral (Lovari and
72
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Apollonio, 1994; Xie, 2006). Breeding introductions were conducted by animal caretakers when females exhibited a tail-up estrous behavior in response to male investigation; mountings and copulations were recorded. 2.4. Fecal extraction and hormone analysis All chemicals were obtained from Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Fecal samples were dried for ∼24 h using a conventional oven at 60 ◦ C and stored at −20 ◦ C until extraction using a fecal extraction method based on Brown et al. (1994). Briefly, samples were thawed at room temperature and 0.1 g (±0.01) of well-mixed dry powdered feces was placed in a glass tube containing 90% ethanol, vigorously shaken in a Multi Pulse vortexer (Glas-Col, Terre Haute, IN) for 30 min, and centrifuged at 2500 × g for 20 min. The extraction was performed twice and the two supernatants were combined, dried down under air in a warm water bath (50 ◦ C) and reconstituted in 1 ml dilution buffer (0.1 M NaPO4 , 0.149 M NaCl, pH 7.0). These extracts were stored at −20 ◦ C until further analysis. Extraction efficiencies were 87% (coefficient of variation (CV) < 15%) for testosterone and 89.2% (CV < 10%) for cortisol based on the recovery of the respective standard added to dried feces before extraction. Androgen and glucocorticoid metabolites were quantified by enzymeimmunoassays (EIA) using antibodies (testosterone polyclonal, R156/7; cortisol polyclonal, R4866) and horseradish peroxidase (HRP)-conjugated tracers obtained from Coralie Munro (University of California, Davis, CA, USA). Antisera cross-reactivities for the testosterone and cortisol EIAs are provided in Young et al. (2001). Each EIA was performed in 96-well plates (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) coated 16–24 h previously with antiserum (50 l; testosterone, 1:8500; cortisol, 1:10,000 dilutions) in coating buffer (0.05 M NaHCO3 , pH 9.6). Standards (50 l; testosterone range, 2.3–600 pg/well; cortisol range, 3.9–1000 pg/well) diluted in assay buffer (0.1 M NaPO4 , 0.149 M NaCl, 0.1% bovine serum albumin, pH 7.0) and samples (50 l; testosterone, 1:30; cortisol, 1:2 dilutions) were combined with HRP (50 l; testosterone, 1:90,000; cortisol, 1:15,000 dilutions) and incubated at room temperature for 2 h (testosterone) or 1 h (cortisol). Plates were washed five times (Biochrom® Anthos Fluido 2 microplate washer, Cambridge, UK) before addition of 100 l substrate (0.4 mM ABTS) to each well. After incubation for 15–45 min, the absorbance was measured at 405 nM (TECAN SunriseTM microplate reader, Salzburg, Austria) until the optical density approached 1.0. The cortisol EIA assay was validated for goral feces by showing that serial dilutions of pooled extracts produced displacement curves parallel to those of the cortisol standard curve (r = 0.9595). Physiological validation of the cortisol EIA was demonstrated by showing a significant increase (100–150%; P < 0.05) in concentrations within 24–48 h after a stressful event (e.g., blood collection, n = 2; semen collection, n = 4). Addition of unlabeled cortisol standard (Sigma Diagnostics Cat. #H4001) to pooled fecal extracts before extraction resulted in a significant (P < 0.05) recovery of mass (y = 0.97x − 0.14, R2 = 0.99). Assay sensitivity was 0.078 ng/ml at 90% binding. Inter-assay CVs
were <15% (n = 96 assays for cortisol and 57 assays for testosterone) based on binding of high (30%) and low (70%) control samples. Samples were re-analyzed if the duplicate CV was >10%; thus, intra-assay CVs were <10%. The testosterone EIA was validated in this study by showing that serial dilutions of pooled fecal extracts produced displacement curves parallel to those of the testosterone standard curve (r = 0.9978). Recovery of added testosterone standard (Steraloids Cat. #A6950) to pooled fecal extracts demonstrated mass recovery (y = 1.01x − 2.12, R2 = 0.99) (P < 0.05). The biological validity of this assay was demonstrated by a clear seasonality in fecal androgen values (see Section 3) and also that concentrations were markedly lower in female goral (overall female mean, 10.15 ± 1.01 ng/g; range, 3.94–17.99 ng/g, n = 5 female gorals and n = 75 samples), which had to be analyzed at a 1:10 dilution compared to 1:30 for males. Assay sensitivities were 0.047 ng/ml for the testosterone and 0.078 ng/ml for the cortisol EIA. All inter-assay CVs were <15% and intra-assay CVs were <10%. Data are expressed as ng/g dry feces. 2.5. High performance liquid chromatography (HPLC) The numbers and relative proportions of immunoactive androgen metabolites in goral feces were determined by reverse phase HPLC (Rudert et al., 2011). HPLC analysis of fecal glucocorticoid metabolites was determined previously (Khonmee et al., unpublished). For assessment of androgen metabolites, five fecal extracts from five animals representing different months were combined, air dried, re-suspended in 1 ml methanol, dried again and stored at −20 ◦ C until analysis. Extract pools were reconstituted with 0.5 ml phosphate buffered saline and filtered through a C-18 spice cartridge (VWR, West Chester, PA), eluted with 5 ml methanol, air dried and resuspended in 300 l methanol spiked with 3 H-testosterone (∼3500 dpm). Androgen metabolites in filtered fecal extracts (55 l) were separated on a Microsorb C-18 column (Reverse Phase MicrosorbTM MV 100 C18, 5 mm diameter particle size; Varian Inc., Woburn, MA) using a gradient of 45% methanol over 90 min (1 ml/fraction; 1 ml/min flow rate). Co-elution of the 3 H-testosterone tracer was determined by adding 100 l of each HPLC fraction to 3 ml of scintillation fluid (Ultima Gold; Packard, Meriden, CT) and counting in a dual-label channel beta scintillation counter (Beckman, Fullerton, CA). The remainder of each fraction (900 l) was evaporated to dryness, reconstituted in 200 l assay buffer and 50 l analyzed by EIA in singlicate. 2.6. Statistical analyses Data are reported as mean ± standard error of the mean (SEM). Androgen and glucocorticoid metabolites were averaged on a weekly and monthly basis, followed by calculations of seasonal means (summer, rainy, winter) and monthly means. Differences in seasonal and monthly means were determined using a generalized least square (GLS) method for repeated measures by R 3.0.0 (R Development Core Team, 2013) with nlme package (Pinheiro et al., 2013). Behavioral data were analyzed on
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
73
Fig. 1. Reverse-phase HPLC separation of immunoreactive androgen metabolites in feces of goral. Immunoreactivity in each fraction was measured with a testosterone EIA. Elution of the 3 H-testosterone reference tracer in HPLC fractions of extracted fecal samples is indicated by the arrow.
a seasonal basis only because of the low (or no) frequency of behaviors for some months. The structure of the covariance pattern for GLS was defined as a compound symmetry. A Bonferroni method was used for the multiple comparison adjustment. Relationships between weekly androgen and glucocorticoid metabolite concentrations and the association of each hormone to the environmental data (average temperature, rainfall, and day length) were analyzed using GLS method because of the correlated data. Pearson’s correlation coefficient analyses were used to determine the correlation in percent binding between serial dilutions of hormone standards and fecal extract dilutions in assay parallelism validation tests. For all statistical tests, the significance level was set at ˛ = 0.05. 3. Results Analysis of HPLC-separated male goral fecal eluates for immunoreactivity using the testosterone EIA revealed the presence of several androgen metabolites, one of which corresponded to radiolabeled testosterone (fractions 31–37; 25% of the immunoactivity) (Fig. 1). Three other more polar metabolite peaks occurred before the elution of testosterone at fractions 8–16 (33%), 17–23 (25%) and 24–28 (12%). Two comparatively smaller, less polar immunoreactive peaks occurred after testosterone at fractions 41–46 (4%) and 50–58 (7%).
A summary of male goral sexual behaviors across seasons is shown in Table 2. In general, males exhibited greater rates of tongue flick, approach and flehmen during the winter season (P < 0.05). Flehmen also was greater in the preceding rainy season (P < 0.05). During the study period, six of the eight male goral (62.5%) bred successfully, based on live births around 7 months later (7.07 ± 0.14 months) in July (n = 2 births), August (n = 1), October (n = 2) and December (n = 1). A number of copulations were observed for each female. Of the six that became pregnant, five copulated once during the conceptive cycle, whereas one bred in August before conceiving after one breeding in December. Two females did not conceive, although breeding was observed (3 and 1 mating each) in May and July, respectively. Overall mean fecal androgen and glucocorticoid metabolite concentrations across season during the study period are shown in Table 3. Average androgen concentrations were greatest in the rainy season and lowest in the summer, with intermediate concentrations in the winter (P < 0.05). Weekly and monthly mean androgen metabolite concentrations across the three seasons are shown in Fig. 2. Compared to the lesser concentrations observed in March, fecal androgens were greater in June to August. Concentrations in July also were greater than those in April and February (P < 0.05). There were positive correlations between fecal androgen metabolite concentrations
Table 2 Mean frequency (±SEM) of male goral sexual behaviors across seasons at Omkoi Wildlife Sanctuary, Chiang Mai, Thailand based on instantaneous scan sampling during 30-min observation periods. Behavior descriptions are based on Xie (2006). Season
Tongue flicka
Approachb
Smell and lickc
Flehmend
Mounte
Summerf Rainyf Winterf
1.32 ± 0.22ab 0.73 ± 0.27a 2.48 ± 0.51b
2.31 ± 0.25a 4.36 ± 2.58ab 4.57 ± 0.38b
1.10 ± 0.38a 1.36 ± 0.54a 2.36 ± 0.47a
0.42 ± 0.10a 1.27 ± 0.47b 1.05 ± 0.17b
0.31 ± 0.31a 0.73 ± 0.73a 1.21 ± 0.53a
Values differ among seasons, different superscript letters (a,b) indicate differences (P < 0.05). a Tongue flick – males flicks tongue between lips toward the female. b Approach – male walks directly toward the female. c Smell and lick – male approaches the female when she is laying down to sniff and lick, sometimes involving naso-genital contact. d Flehmen – male lifts head and curls the lips after sniffing female or her urine. e Mount – male rises on hind legs and rests forelegs and chest on the female. f Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
74
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Fig. 2. (a) Monthly mean (±SEM) androgen metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center. Values differ among seasons, different letters (a,b,c,d) indicate differences (P < 0.05). (b) Longitudinal mean (±SEM) androgen metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center.
and temperature (y = −12.88 + 11.19 × average temperature, P < 0.05) and day length (y = −134.49 + 31.75 × day length, P < 0.05) across the year. Mean glucocorticoid metabolite concentrations were less in the summer (P < 0.05), with no difference between rainy and winter seasons (Table 3), a pattern that is discernible in Fig. 3. Similar to androgens and compared to March, glucocorticoid concentrations were elevated in June to August and again in November to January (i.e., the rainy and winter seasons) (P < 0.05). In contrast to androgens, there were no correlations between glucocorticoid metabolite concentrations and mean temperature and day length. However, glucocorticoids were negatively correlated with rainfall (y = 21.57 − 0.24 × rainfall, P < 0.05). There also was a positive relationship between androgen and glucocorticoid metabolite concentrations (y = 64.82 + 8.82 × cortisol, P < 0.05) for the year. 4. Discussion This is the first study to characterize steroid hormone profiles in male goral, and confirmed the influence of seasonality on testicular and adrenal function. Overall concentrations of fecal androgen and glucocorticoid metabolites were greater in the rainy season and remained elevated throughout most of the winter before decreasing in the summer. The increase corresponded with successful copulations starting in July in the Omkoi males. Fecal androgen concentrations were positively correlated with temperature and day length, although glucocorticoids were not. Overall, results indicate that testicular and adrenal
steroidogenic activity in captive-held goral in Thailand follow a modest, but significant seasonal pattern, and at least for testicular activity may be initiated in part by subtle changes in photoperiod. A previous study validated the cortisol EIA for quantifying glucocorticoid metabolites in goral feces, including showing that native cortisol is excreted in appreciable amounts (17.5% of total immunoreactivity; Khonmee et al., unpublished). This study found that native testosterone also is excreted in goral feces (25%) in addition to other primarily more polar metabolites. Other species excrete native testosterone in feces in varying proportions [hyena (Crocuta crocuta), Dloniak et al., 2004); rhinoceros (Ceratotherium simum simum), Kretzschmar et al., 2004; bison (Bison bison bison),Mooring et al., 2004; chinchilla (Chinchilla lanigera), Busso et al., 2005], although not all do (felids, Brown et al., 1996; primates, Möhle et al., 2002). Using the EIA in the present study, the pattern of goral androgen production was similar to that of other seasonal ungulates (reindeer (Rangifer tarandus L.), Mossing and Damber, 1981; white-tailed deer (Odocoileus virginianus), Bubenik et al., 1987; wild and domestic sheep, Lincoln et al., 1990; Pelletier et al., 2003; impala (Aepyceros melampus), Brown et al., 1991; red deer (Cervus elaphus), Freudenberger et al., 1993; bison, Mooring et al., 2004), including several caprine species (Pérez and Mateos, 1995; Todini et al., 2007; Zarazaga et al., 2010; Polat et al., 2011), where concentrations increase preceding or in conjunction with rut or breeding activity. Chinese goral are reported to be distinctly seasonal with breeding occurring in the winter (October–January:
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
75
Fig. 3. (a) Monthly mean (±SEM) glucocorticoid metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center. Values differ among seasons, different letters (a,b,c,d) indicate differences (P < 0.05). (b) Longitudinal mean (±SEM) glucocorticoid metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center.
Kanbunjong, 1993; November–December: Lekagul and McNeely, 1988), a time frame that agrees with that for captive and wild Himalayan and red goral (Lovari and Apollonio, 1994; Xie, 2006; Grubb, 2005; Duckworth and MacKinnon, 2008). The closely related serow species (now in the genus Capricornis) also breeds primarily between November and January in situ (Duckworth et al., 2008). And sika deer (Cervus nippon), another Asian species, exhibit increased excreted gonadal steroids in both sexes during October–January (Yamauchi et al., 1997; Hamasaki et al., 2001). Thus, many if not most seasonal ungulates in Asia, including Thailand, breed toward the end of the year, during the cooler months of winter. At Omkoi however, androgen metabolites began to increase several months earlier, in July, and remained elevated throughout both rainy and winter season months. Females also displayed estrus earlier in the year based on observations of tail-up behaviors, which were accompanied by conceptive matings as early as 21 July. Thus, it appears that captive goral in Thailand, at least at Omkoi, breed earlier than wild counterparts, and exhibit an overall longer breeding season. This could be a captivity effect; some seasonal ungulates undergo changes in reproductive patterns, becoming less seasonal, non-seasonal or exhibiting extended breeding seasons ex situ (Skinner et al., 2002; Zerbe et al., 2012; Kaumanns et al., 2013). Reproductive seasonality is generally driven by two forces, alone or in combination: photoperiod and resource availability (Bronson, 1989; Kaumanns et al., 2013). There
were moderate differences across seasons in temperature and day length at Omkoi, about 3.5 ◦ C and 1.5 h from shortest to longest, respectively, and these were correlated, modestly though significantly, with fecal androgens. Thus, reproductive seasonality in goral may be regulated in part by photoperiod changes, a finding that has been well documented in other ungulates (see review, Zerbe et al., 2012). The Omkoi Wildlife Breeding Center is located inside a wildlife sanctuary where wild goral live, so the assumption was that captive and wild animals are exposed to similar environmental and photoperiodic conditions. However, there were elevation and likely climatic differences; wild goral prefer rocky crags at high elevations (1400–1929 m; Chaiyarat et al., 1999), whereas the elevation of the captive breeding center was much lower at only ∼500 m.
Table 3 Mean (±SEM) fecal androgen and glucocorticoid metabolite concentrations in male goral across seasons at Omkoi Wildlife Sanctuary, Chiang Mai, Thailand. Season
Androgen metabolites (ng/g)
Glucocorticoid metabolites (ng/g)
Summera Rainya Wintera
195.48 ± 8.23a 289.82 ± 9.18c 224.09 ± 11.97b
15.30 ± 0.48a 22.10 ± 0.72b 21.98 ± 0.98b
Values differ among seasons, different supercript letters (a,b,c) indicate differences (P < 0.05). a Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
76
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Another possibility for the early onset of breeding activity could be related to resources. Omkoi animals are fed natural forage, but also are provisioned with a pelleted concentrate daily. Thus, body condition remained relatively constant throughout the year compared to wild counterparts. Others have demonstrated the importance of good nutrition and body condition on enhancing reproductive function; for example, in both wild (Albon et al., 1986) and captive (Heydon et al., 1992; Carrion et al., 2008; GasparLópez et al., 2010) deer. So, males at Omkoi may attain a functional breeding state sooner than expected because of better overall body condition earlier in the season. Types of sexual behaviors of Chinese goral were similar to those reported for the Himalayan (Lovari and Apollonio, 1994) and red (Xie, 2006) goral, and included approach, smell and lick, tongue flick, and flehmen. At Omkoi, the frequency of behaviors was generally greater in the winter, and significantly for tongue flick and approach. Interestingly, this was after most of the females had conceived in this study. The flehmen response, however, was greater in both the rainy and winter seasons, when most of the conceptions occurred. Approach was the behavior most frequently observed at Omkoi, whereas in red goral smell and lick were most common (Xie, 2006). In Himalayan goral, the most common courtship behaviors were approach, following, mount and naso-nasal contact (Lovari and Apollonio, 1994). Both of these previous studies were conducted during the purported breeding season only (October for Lovari and Apollonio, 1994; September–November for Xie, 2006), however, so behaviors outside the breeding season were not documented. Still, considering these reports, it was surprising to find that males at Omkoi exhibited sexual behaviors throughout the year, as did females (e.g., tailup). However, greater androgen metabolite concentrations were observed during the months of active breeding, not unlike that reported for other ungulate species (Li et al., 2000, 2001; Imwalle et al., 2002; Mooring et al., 2004). Although there were no correlations between fecal glucocorticoid metabolite concentrations and temperature or day length, as with androgens, overall fecal glucocorticoid metabolites exhibited a clear seasonality, with concentrations being greater during the rainy and winter seasons as compared to the summer. Other ungulate species exhibit increased glucocorticoid production during winter [whitetailed deer (Odocoileus virginianus), Bubenik et al., 1983; Mccoy and Ditchkoff, 2012; pygmy goat (Capra hircus), Howland et al., 1985; mule deer (Odocoileus hemionus), Saltz and White, 1991] or late fall (bison, Mooring et al., 2006) seasons. Others, however, have no distinct seasonal glucocorticoid fluctuations, such as axis deer (Axis axis) (Bubenik and Brown, 1989), Eld’s deer (Cervus eldi thamin) (Monfort et al., 1993), pudu (Pudu pudu) (Reyes et al., 1997) and reindeer (Rangifer tarandus) (Bubenik et al., 1998). Differences in adrenal patterns may be species specific; however, it often is difficult to determine if increases in concentrations of glucocorticoids related to reproduction and/or androgen production are due to environmental or physiological effects when the two change in concert, as observed in goral. Although increased glucocorticoid concentrations during some types of stress can suppress testicular function and correlate negatively with
androgen secretion (baboon, Sapolsky, 1985; rat, Orr and Mann, 1992; guinea pig, Fenske, 1997; mice, Dong et al., 2004), co-production of androgens and glucocorticoids is observed during the normal breeding season in many ungulate species as noted above, including goral. 5. Conclusion This is the first study to assess male endocrine function in goral and to ascertain seasonal variation in testicular and adrenal steroidogenic function with greater activity in the rainy season and winter. Although wild goral breed primarily during the winter months, results of the present study indicate that captive males can be reproductively functional as early as July, and remain fertile through December. The function of enhanced adrenal activity during the rainy season and winter in male goral is not clear, but may be due to environmental conditions or breeding activity or both. Given that resources for goral at Omkoi are consistent throughout the year, reproduction may be regulated to a great extent by photoperiod. However, the early onset and extended breeding season at Omkoi suggests that maintaining consistently good body condition throughout the year may have a positive influence on initiation of spermatogenic function and reproductive behavior. Alternatively, being housed at a comparatively lower elevation compared to wild goral could have altered physiological responses to season. Knowledge of how endocrine function changes seasonally in goral can be used to enhance captive breeding, such as determining optimal times for semen collection and genome resource banking in conjunction with the use of ART procedures. Although semen analyses have yet to be been conducted, it appears that functional spermatozoa probably can be collected within 2–3 months after the start of the rainy season through the end of winter. Acknowledgments This study was supported by National Research Council of Thailand (NRCT) and the Graduate School of Chiang Mai University. The authors extend their gratitude to the staff of the Zoological Park Organization of Thailand, and Omkoi Wildlife Sanctuary for their assistance in sample collection. We are also grateful to Ms. Nicole Presley at the Smithsonian Conservation Biology Institute, and staff of the endocrinology lab at the Faculty of Veterinary Medicine, Chiang Mai University (Ms. Manisorn Tuantammarak, Ms. Patharanun Wongchai, and Mr. P allop Tankaew) for technical support. References Albon, S.D., Mitchell, B., Huby, B.J., Brown, D., 1986. Fertility in female red deer (Cervus elaphus): the effects of body composition, age and reproductive status. J. Zool. 209, 447–460. Bronson, F.H., 1989. Mammalian Reproductive Biology. University of Chicago Press, Chicago. Brown, J.L., Terio, K.A., Graham, L.H., 1996. Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids. Zoo Biol. 15, 425–434. Brown, J.L., Wasser, S.K., Wildt, D.E., Graham, L.H., 1994. Comparative aspects of steroid hormone metabolism and ovarian activity in felids, measured noninvasively in feces. Biol. Reprod. 51, 776–786.
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78 Brown, J.L., Wildt, D.E., Raath, J.R., de Vos, V., Janssen, D.L., Citino, S.B., Howard, J.G., Bush, M., 1991. Seasonal variation in pituitary-gonadal function in free-ranging impala (Aepyceros melampus). J. Reprod. Fertil. 93, 497–505. Bubenik, G.A., Brown, R.D., 1989. Seasonal levels of cortisol, triiodothyronine and thyroxine in male axis deer. Comp. Biochem. Physiol. A: Physiol. 92, 499–503. Bubenik, G.A., Bubenik, A.B., Schams, D., Leatherland, J.F., 1983. Circadian and circannual rhythms of LH, FSH, testosterone, prolactin, cortisol, T3 and T4 in plasma of mature, male white-tailed deer. Comp. Biochem. Physiol. A: Physiol. 76, 37–45. Bubenik, G.A., Pomerantz, D.K., Schams, D., Smith, P.S., 1987. The role of androstenedione and testosterone in the reproduction and antler growth of a male white-tailed deer. Acta Endocrinol. 114, 147–152. Bubenik, G.A., Schams, D., White, R.G., Rowell, J., Blake, J., Bartos, L., 1998. Seasonal levels of metabolic hormones and substrates in male and female reindeer (Rangifer tarandus). Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 120, 307–315. Busso, J.M., Ponzio, M.F., Dabbene, V., Cuneo, M.F., de Ruiz, R.D., 2005. Assessment of urine and fecal testosterone metabolite excretion in Chinchilla lanigera males. Anim. Reprod. Sci. 86, 339–351. Carrion, D., Garcıa, A.J., Gaspar-Lopez, E., Landete-Castillejos, T., Gallego, L., 2008. Development of body condition and its effect on calf birth weight and milk production. J. Exp. Zool. 309A, 1–10. Chaiyarat, R., Laohajinda, W., Kutintara, U., Nabhitabhata, J., 1999. Ecology of the goral (Naemorhedus goral) in Omkoi Wildlife Sanctuary Thailand. Nat. Hist. Bull. SIAM Soc. 47, 191–205. Conde, D.A., Flesness, N., Colchero, F., Jones, O.R., Scheuerlein, A., 2011. An emerging role of zoos to conserve biodiversity. Science 331, 1390–1391. Dloniak, S.M., French, J.A., Place, N.J., Weldele, M.L., Glickman, S.E., Holekamp, K.E., 2004. Non-invasive monitoring of fecal androgens in spotted hyenas (Crocuta crocuta). Gen. Comp. Endocrinol. 135, 51–61. Dong, Q., Salva, A., Sottas, C.M., Niu, E., Holmes, M., Hardy, M.P., 2004. Rapid glucocorticoid mediation of suppressed testosterone biosynthesis in male mice subjected to immobilization stress. J. Androl. 25, 973–981. Duckworth, J.W., Steinmetz, R., Chaiyarat, R., 2008. Naemorhedus griseus. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 [WWW Document]. http://www.iucnredlist.org (accessed 23.4.13). Duckworth, J.W., MacKinnon, J., 2008. Naemorhedus caudatus. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2013.1 [WWW Document]. http://www.iucnredlist.org/details/14294/0 (accessed 8.8.13). Fenske, M., 1997. Role of cortisol in the ACTH-induced suppression of testicular steroidogenesis in guinea pigs. J. Endocrinol. 154, 407–414. Freudenberger, D.O., Wilson, P.R., Barry, T.N., Sun, Y.X., Purchas, R.W., Trigg, T.E., 1993. Effects of immunization against GnRH upon body growth, voluntary food intake and plasma hormone concentration in yearling red deer stags (Cervus elaphus). J. Agric. Sci. 121, 381–388. Gaspar-López, E., Landete-Castillejos, T., Estevez, J., Ceacero, F., Gallego, L., García, A., 2010. Biometrics, testosterone, cortisol and antler growth cycle in Iberian red deer stags (Cervus elaphus hispanicus). Reprod. Domest. Anim. 45, 243–249. Grubb, P., 2005. Order Artiodactyla. In: Wilson, D.E., Reeder, D.M. (Eds.), Mammal Species of the World: A Taxonomic and Geographic Reference. , third ed. The Johns Hopkins University Press, Baltimore, pp. 637–722. Hamasaki, S., Yamauchi, K., Ohki, T., Murakami, M., Takahara, Y., Takeuchi, Y., Mori, Y., 2001. Comparison of various reproductive status in sika deer (Cervus nippon) using fecal steroid analysis. J. Vet. Med. Sci. 63, 195–198. Heydon, M.J., Loudon, A.S.I., Milne, J.A., Brinklow, K.R., 1992. Influence of plane of nutrition on seasonal changes in food intake and reproduction in grazing red deer. In: Brown, R.D. (Ed.), Antler Development in Cervidae. Caesar Kleberg Wildlife Research Institute, Kingsville, pp. 279–284. Howland, B.E., Sanford, L.M., Palmer, W.M., 1985. Changes in serum levels of LH FSH, prolactin, testosterone, and cortisol associated with season and mating in male pygmy goats. J. Androl. 6, 89–96. Huber, S., Palme, R., Arnold, W., 2003. Effects of season, sex, and sample collection on concentrations of fecal cortisol metabolites in red deer (Cervus elaphus). Gen. Comp. Endocrinol. 130, 48–54. Imwalle, D.B., Daxenberger, A., Schillo, K.K., 2002. Effects of melengestrol acetate on reproductive behavior and concentrations of LH andtestosterone in bulls. J. Anim. Sci. 80, 1059–1067. Kanbunjong, S., 1993. Behavior of Goral (Naemorhaedus griseus) in Captivity. Chiang Mai University, Chiang Mai. Kretzschmar, P., Gansloßer, U., Dehnhard, M., 2004. Relationship between androgens, environmental factors and reproductive behavior in male
77
white rhinoceros (Ceratotherium simum simum). Horm. Behav. 45, 1–9. Lekagul, B., McNeely, J.A., 1988. Mammals of Thailand. Darnsutha Press, Bangkok. Li, C., Jiang, Z., Fang, J., Jiang, G., Ding, Y., Shen, H., Xu, A., 2000. Relationship between reproductive behavior and fecal steroid in milu (Elaphurus davidianus). Acta Theriol. Sin. 20, 88–100. Li, C., Jiang, Z., Jiang, G., Fang, J., 2001. Seasonal changes of reproductive behavior and fecal steroid concentrations in Père David’s deer. Horm. Behav. 40, 518–525. Lincoln, G.A., Lincoln, C.E., McNeilly, A.S., 1990. Seasonal cycles in the blood plasma concentration of FSH, inhibin and testosterone, and testicular size in rams of wild, feral and domesticated breeds of sheep. J. Reprod. Fertil. 88, 623–633. Lovari, S., Apollonio, M., 1994. On the rutting behaviour of the Himalayan goral Nemorhaedus goral (Hardwicke, 1825). J. Ethol. 12, 25–34. Mccoy, J.C., Ditchkoff, S.S., 2012. Patterns of fecal hormones in a fenced population of white-tailed deer. Wildl. Soc. Bull. 36, 641–646. Millspaugh, J.J., Washburn, B.E., 2004. Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation. Gen. Comp. Endocrinol. 138, 189–199. Möhle, U., Heistermann, M., Palme, R., Hodges, J.K., 2002. Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates. Gen. Comp. Endocrinol. 129, 135–145. Monfort, S.L., Brown, J.L., Wildt, D.E., 1993. Episodic and seasonal rhythms of cortisol secretion in male Eld’s deer (Cervus eldi thamin). J. Endocrinol. 138, 41–49. Mooring, M.S., Patton, M.L., Lance, V.A., Hall, B.M., Schaad, E.W., Fetter, G.A., Fortin, S.S., McPeak, K.M., 2006. Glucocorticoids of bison bulls in relation to social status. Horm. Behav. 49, 369–375. Mooring, M.S., Patton, M.L., Lance, V.A., Hall, B.M., Schaad, E.W., Fortin, S.S., Jella, J.E., McPeak, K.M., 2004. Fecal androgens of bison bulls during the rut. Horm. Behav. 46, 392–398. Mossing, T., Damber, J.E., 1981. Rutting behavior and androgen variation in reindeer (Rangifer tarandus L.). J. Chem. Ecol. 7, 377–389. Orr, T.E., Mann, D.R., 1992. Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats. Horm. Behav. 26, 350–363. Patton, M.L., Aubrey, L., Edwards, M., Rieches, R., Zuba, J., Lance, V.A., 2000. Successful contraception in a herd of Chinese goral (Nemorhaedus goral arnouxianus) with melengestrol acetate. J. Zoo Wildl. Med. 31, 228–230. Pelletier, F., Bauman, J., Festa-Bianchet, M., 2003. Fecal testosterone in bighorn sheep (Ovis canadensis): behavioural and endocrine correlates. Can. J. Zool. 81, 1678–1684. Pérez, B., Mateos, E., 1995. Seasonal variations in plasma testosterone ˜ bucks. Small Rumin. Res. 15, 155–162. levels in Verata and Malaguena Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., the R Development Core Team, 2013. nlme: Linear and Nonlinear Mixed Effects Models. In: R Package Version 3.1-110. R Development Core Team, 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3-900051-07-0 [WWW Document] http://www.R-project.org/ (accessed 1.7.13). Rabinowitz, A., Khaing, S.T., 1998. Status of selected mammal species in North Myanmar. Oryx 32, 201–208. Reyes, E., Bubenik, G.A., Lobos, A., Schams, D., Bartos, L., 1997. Seasonal levels of cortisol. IGF-1 and triiodothyronine in adultmale pudu (Pudu pudu). Fol. Zool. 46, 109–116. Rudert, S., Brown, J., Gansloßer, l., Möbius, U., Songsasen, G.N., 2011. Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides). Zoo Biol. 30, 134–148. Saltz, D., White, G.C., 1991. Urinary cortisol and urea nitrogen responses to winter stress in mule deer. J. Wildl. Manage. 55, 1. Sapolsky, R.M., 1985. Stress-induced suppression of testicular function in the wild baboon: role of glucocorticoids. Endocrinology 116, 2273–2278. Skinner, J.D., Moss, D.G., Skinner, D.C., 2002. Inherent seasonality in the breeding seasons of African mammals: evidence from captive breeding. Trans. R. Soc. S. Afr. 57, 25–33. Thai Meteorological Department [WWW Document], 2013. http://www.tmd.go.th/en/ (accessed 24.4.13). Todini, L., Malfatti, A., Terzano, G.M., Borghese, A., Pizzillo, M., Debenedetti, A., 2007. Seasonality of plasma testosterone in males of four Mediterranean goat breeds and in three different climatic conditions. Theriogenology 67, 627–631.
78
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Xie, Y., 2006. Primary observations on rutting behavior of the captive red goral. Zoo Biol. 25, 117–123. Yamauchi, K., Hamasaki, S., Takeuchi, Y., Mori, Y., 1997. Assessment of reproductive status of sika deer by fecal steroid analysis. J. Reprod. Dev. 43, 221–226. Young, K.M., Brown, J.L., Goodrowe, K.L., 2001. Characterization of reproductive cycles and adrenal activity in the black-footed ferret (Mustela nigripes) by fecal hormone analysis. Zoo Biol. 20, 517–536.
Zarazaga, L.A., Gatica, M.C., Celi, I., Guzmán, J.L., Malpaux, B., 2010. Effect of artificial long days and/or melatonin treatment on the sexual activity of Mediterranean bucks. Small Rumin. Res. 93, 110–118. Zerbe, P., Clauss, M., Codron, D., Bingaman Lackey, L., Rensch, E., Streich, J.W., Hatt, J.-M., Müller, D.W.H., 2012. Reproductive seasonality in captive wild ruminants: implications for biogeographical adaptation, photoperiodic control, and life history. Biol. Rev. Camb. Philos. Soc. 87, 965–990.
Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Seasonality of fecal androgen and glucocorticoid metabolite excretion in male goral (Naemorhedus griseus) in Thailand Jaruwan Khonmee a,∗ , Janine L. Brown b , Suvichai Rojanasthien a , Dissakul Thumasanukul c , Adisorn Kongphoemphun c , Boripat Siriaroonrat d , Wanlaya Tipkantha d , Veerasak Punyapornwithaya a , Chatchote Thitaram a a b c d
Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Center for Species Survival, Smithsonian Conservation Biology Institute, Front Royal, VA 22630, USA Omkoi Wildlife Sanctuary, Department of National Park, Wildlife and Plant Conservation, Omkoi, Chiang Mai 50310, Thailand Conservation Research and Education Division, Zoological Park Organization, Dusit, Bangkok 10300, Thailand
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 31 January 2014 Accepted 7 February 2014 Available online 19 February 2014
Keywords: Goral Glucocorticoid metabolites Androgen metabolites Non-invasive hormone monitoring Seasonality
a b s t r a c t There is no information on the endocrinology of Chinese goral (Naemorhedus griseus), a high priority species for captive breeding and reintroduction in Thailand. This study characterized fecal androgen and glucocorticoid metabolites in male goral at Omkoi Wildlife Sanctuary to investigate seasonal relationships. Fecal samples were collected 3 days/week for 1 year from eight adult males. Mean androgen metabolite concentrations were greater (P < 0.05) during the rainy season (289.82 ± 9.18 ng/g) and winter (224.09 ± 11.97 ng/g) compared to the summer (195.48 ± 8.23 ng/g), and were related to breeding activity. A similar pattern was observed for glucocorticoid concentrations (22.10 ± 0.72 ng/g compared to 21.98 ± 0.98 ng/g compared to 15.30 ± 0.48 ng/g), respectively, and this resulted in a positive correlation between the two hormones (P < 0.05). There also were positive correlations between fecal androgen metabolite concentrations and temperature (P < 0.05) and day length (P < 0.05). In summary, this is the first study to assess endocrine function in male goral, and results showed seasonal variation in testicular and adrenal steroidogenic function, with greater activity in the rainy season and winter. Given that resources for captive male goral are consistent throughout the year, reproduction may be regulated primarily by photoperiod in this species. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chinese goral (Naemorhedus griseus) are small ungulates with a goat-like appearance that inhabit steep rocky terrain throughout Burma, China, India, Thailand and Vietnam (Rabinowitz and Khaing, 1998; Patton et al., 2000;
∗ Corresponding author. Tel.: +66 53 948 046; fax: +66 53 948 065. E-mail addresses: [email protected], [email protected] (J. Khonmee). http://dx.doi.org/10.1016/j.anireprosci.2014.02.008 0378-4320/© 2014 Elsevier B.V. All rights reserved.
Duckworth et al., 2008). These animals are listed as ‘Vulnerable’ on the IUCN Red List (Duckworth et al., 2008) and are one of 15 protected species under the Wild Animal Reservation and Protection Act of Thailand (Chaiyarat et al., 1999). Goral live in small family groups, with males defending territories of ∼25 ha (Duckworth et al., 2008). In Thailand, this species is found exclusively in protected areas of Northern Thailand, in steep steppe areas and mountainous plateaus (Chaiyarat et al., 1999). The size of wild goral populations is unknown, but numbers are believed to be decreasing due to habitat loss and hunting by local people for meat, fur and
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
traditional medicine (Duckworth et al., 2008). As a result, this is a priority species of the Zoological Parks Organization of Thailand for re-introduction and captive breeding efforts. There are about 100 goral held among three captive facilities in Thailand (Omkoi Wildlife Sanctuary, Chiang Mai Night Safari and Chiang Mai Zoo). Animals are breeding at the first two facilities; however, reproduction is suboptimal and populations are not self-sustaining. There is interest in developing assisted reproductive technologies (ART) for this species, for example artificial insemination and embryo transfer, to supplement captive breeding efforts and facilitate genetic management. Inbreeding is a particular concern as routine transferring of males between facilities for breeding has not been a logistically successful strategy. Zoos and captive breeding are having an increasingly important role in conservation efforts of many threatened species (Conde et al., 2011), but success is predicated on a thorough knowledge of species biology, something that is lacking for goral. Previous studies have focused on observations of sexual behavior, particularly rutting activities in captive male and female Himalayan (Neamorhedus goral; Lovari and Apollonio, 1994) and red (Naemorhedus cranbrooki; Xie, 2006) goral. All indications are that these animals are seasonal, with maximal activity in the winter months. In Chinese goral, breeding has been reported to occur between November and December in the wild (Lekagul and McNeely, 1988) and between October and January at the Choengdoi Suthep Nature and Wildlife Education Centre (Kanbunjong, 1993). However, there are no endocrine data to confirm this seasonality in Chinese or any other goral species. Given the limited amount of reproductive information, and the need to determine optimal times for scheduling ART procedures, studies to characterize androgen and glucocorticoid activity in male goral are warranted. Most studies of wildlife utilize non-invasive steroid metabolite monitoring to assess endocrine function as an alternative to blood sampling (Huber et al., 2003; Millspaugh and Washburn, 2004). Because hormonal metabolites in feces represent an accumulation over time, these metabolites can more accurately reflect average values (Pelletier et al., 2003). Fecal androgen metabolite analyses have been used to characterize seasonal testicular activity in ungulate species (Yamauchi et al., 1997; Li et al., 2001; Pelletier et al., 2003; Mooring et al., 2004).
Table 1 Mean (±SEM) daily temperature, rainfall and day length across seasons at Omkoi Wildlife Sanctuary during the study period. Data were obtained from The Northern Meteorological Center, Meteorological Department, Chiang Mai, Thailand. Season
Average temperature (◦ C)
Rainfall (mm)
Day length (h)
Summera Rainya Wintera
23.47 ± 2.30b 24.59 ± 1.85c 19.77 ± 1.69a
2.92 ± 0.29b 5.19 ± 0.39c 1.40 ± 0.12a
12.28 ± 0.03b 12.76 ± 0.02c 11.20 ± 0.01a
Values differ among seasons, different superscript letters (a,b,c) indicate differences (P < 0.05). a Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
71
Thus, the objectives of the present study were to (1) determine the influence of season on testicular steroidogenic function in Chinese goral by assessing androgen metabolite concentrations in feces, and (2) examine seasonal relationships between fecal androgen and glucocorticoid metabolite excretion and sexual behavior throughout the year. The ultimate goal is to develop appropriate management strategies that can be implemented to propagate a threatened species that is of national importance to Thailand. 2. Materials and methods 2.1. Animals and samples collection All animal procedures were approved by the Animal Ethic Committee Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand (S22/2553). Eight captive-born male goral (average age, 5.29 ± 0.24 years) were housed individually in outdoor enclosures (6 m × 9 m × 3 m) adjacent to females at Omkoi Wildlife Breeding Center in Chiang Mai, Thailand (17◦ 48 4 N, 98◦ 21 31 E). All enclosures had dirt floors, an open shelter and several natural trees for shade. Animals were exposed to natural light, and fed roughage (Panicum grass; Brachiaria mutica), plus concentrate (500 g, Betagro 009 cattle finisher pellet (12% protein, 2% fat, 13% fiber, 13% moisture); Betagro Company Limited, Thailand) and natural leaves (little yellow star, Melampodium divaricatum) once daily with unlimited access to fresh water. Feces were removed every evening, and freshly defecated feces (∼30 g) were collected between 0830 and 0930 h every morning from each male goral 3 days/week for 1 year. Samples were stored at −20 ◦ C until analysis. Enclosures were configured in two rows of eight adjoining pens, alternating between male and female, with a common corridor in between. 2.2. Environmental data There are three defined seasons in Thailand: summer (February 16 to May 15), rainy season (May 16 to October 15) and winter (October 16 to February 15). Information on average temperature (◦ C), amount of rainfall (mm/day) and day length (h) in each month of the study was obtained from The Northern Meteorological Center, Meteorological Department, Ministry of Information and Communication Technology, Chiang Mai, Thailand (Thai Meteorological Department, 2013). There were seasonal differences in average temperature, rainfall and day length at Omkoi Wildlife Sanctuary, being greatest in the rainy season and lowest in the winter (P < 0.05) (Table 1). 2.3. Behavioral data Every morning of the study, each male goral was placed in the common area, which permitted visual and olfactory access to females. Quantitative behavioral data were collected by instantaneous scan sampling during daily 30-min observation sessions. Male sexual behaviors were recorded and included tongue flick, approach to female, smell, lick and flehmen as described for red goral (Lovari and
72
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Apollonio, 1994; Xie, 2006). Breeding introductions were conducted by animal caretakers when females exhibited a tail-up estrous behavior in response to male investigation; mountings and copulations were recorded. 2.4. Fecal extraction and hormone analysis All chemicals were obtained from Sigma Chemical Company (St. Louis, MO), unless otherwise stated. Fecal samples were dried for ∼24 h using a conventional oven at 60 ◦ C and stored at −20 ◦ C until extraction using a fecal extraction method based on Brown et al. (1994). Briefly, samples were thawed at room temperature and 0.1 g (±0.01) of well-mixed dry powdered feces was placed in a glass tube containing 90% ethanol, vigorously shaken in a Multi Pulse vortexer (Glas-Col, Terre Haute, IN) for 30 min, and centrifuged at 2500 × g for 20 min. The extraction was performed twice and the two supernatants were combined, dried down under air in a warm water bath (50 ◦ C) and reconstituted in 1 ml dilution buffer (0.1 M NaPO4 , 0.149 M NaCl, pH 7.0). These extracts were stored at −20 ◦ C until further analysis. Extraction efficiencies were 87% (coefficient of variation (CV) < 15%) for testosterone and 89.2% (CV < 10%) for cortisol based on the recovery of the respective standard added to dried feces before extraction. Androgen and glucocorticoid metabolites were quantified by enzymeimmunoassays (EIA) using antibodies (testosterone polyclonal, R156/7; cortisol polyclonal, R4866) and horseradish peroxidase (HRP)-conjugated tracers obtained from Coralie Munro (University of California, Davis, CA, USA). Antisera cross-reactivities for the testosterone and cortisol EIAs are provided in Young et al. (2001). Each EIA was performed in 96-well plates (Nunc Maxisorp, Fisher Scientific, Pittsburgh, PA) coated 16–24 h previously with antiserum (50 l; testosterone, 1:8500; cortisol, 1:10,000 dilutions) in coating buffer (0.05 M NaHCO3 , pH 9.6). Standards (50 l; testosterone range, 2.3–600 pg/well; cortisol range, 3.9–1000 pg/well) diluted in assay buffer (0.1 M NaPO4 , 0.149 M NaCl, 0.1% bovine serum albumin, pH 7.0) and samples (50 l; testosterone, 1:30; cortisol, 1:2 dilutions) were combined with HRP (50 l; testosterone, 1:90,000; cortisol, 1:15,000 dilutions) and incubated at room temperature for 2 h (testosterone) or 1 h (cortisol). Plates were washed five times (Biochrom® Anthos Fluido 2 microplate washer, Cambridge, UK) before addition of 100 l substrate (0.4 mM ABTS) to each well. After incubation for 15–45 min, the absorbance was measured at 405 nM (TECAN SunriseTM microplate reader, Salzburg, Austria) until the optical density approached 1.0. The cortisol EIA assay was validated for goral feces by showing that serial dilutions of pooled extracts produced displacement curves parallel to those of the cortisol standard curve (r = 0.9595). Physiological validation of the cortisol EIA was demonstrated by showing a significant increase (100–150%; P < 0.05) in concentrations within 24–48 h after a stressful event (e.g., blood collection, n = 2; semen collection, n = 4). Addition of unlabeled cortisol standard (Sigma Diagnostics Cat. #H4001) to pooled fecal extracts before extraction resulted in a significant (P < 0.05) recovery of mass (y = 0.97x − 0.14, R2 = 0.99). Assay sensitivity was 0.078 ng/ml at 90% binding. Inter-assay CVs
were <15% (n = 96 assays for cortisol and 57 assays for testosterone) based on binding of high (30%) and low (70%) control samples. Samples were re-analyzed if the duplicate CV was >10%; thus, intra-assay CVs were <10%. The testosterone EIA was validated in this study by showing that serial dilutions of pooled fecal extracts produced displacement curves parallel to those of the testosterone standard curve (r = 0.9978). Recovery of added testosterone standard (Steraloids Cat. #A6950) to pooled fecal extracts demonstrated mass recovery (y = 1.01x − 2.12, R2 = 0.99) (P < 0.05). The biological validity of this assay was demonstrated by a clear seasonality in fecal androgen values (see Section 3) and also that concentrations were markedly lower in female goral (overall female mean, 10.15 ± 1.01 ng/g; range, 3.94–17.99 ng/g, n = 5 female gorals and n = 75 samples), which had to be analyzed at a 1:10 dilution compared to 1:30 for males. Assay sensitivities were 0.047 ng/ml for the testosterone and 0.078 ng/ml for the cortisol EIA. All inter-assay CVs were <15% and intra-assay CVs were <10%. Data are expressed as ng/g dry feces. 2.5. High performance liquid chromatography (HPLC) The numbers and relative proportions of immunoactive androgen metabolites in goral feces were determined by reverse phase HPLC (Rudert et al., 2011). HPLC analysis of fecal glucocorticoid metabolites was determined previously (Khonmee et al., unpublished). For assessment of androgen metabolites, five fecal extracts from five animals representing different months were combined, air dried, re-suspended in 1 ml methanol, dried again and stored at −20 ◦ C until analysis. Extract pools were reconstituted with 0.5 ml phosphate buffered saline and filtered through a C-18 spice cartridge (VWR, West Chester, PA), eluted with 5 ml methanol, air dried and resuspended in 300 l methanol spiked with 3 H-testosterone (∼3500 dpm). Androgen metabolites in filtered fecal extracts (55 l) were separated on a Microsorb C-18 column (Reverse Phase MicrosorbTM MV 100 C18, 5 mm diameter particle size; Varian Inc., Woburn, MA) using a gradient of 45% methanol over 90 min (1 ml/fraction; 1 ml/min flow rate). Co-elution of the 3 H-testosterone tracer was determined by adding 100 l of each HPLC fraction to 3 ml of scintillation fluid (Ultima Gold; Packard, Meriden, CT) and counting in a dual-label channel beta scintillation counter (Beckman, Fullerton, CA). The remainder of each fraction (900 l) was evaporated to dryness, reconstituted in 200 l assay buffer and 50 l analyzed by EIA in singlicate. 2.6. Statistical analyses Data are reported as mean ± standard error of the mean (SEM). Androgen and glucocorticoid metabolites were averaged on a weekly and monthly basis, followed by calculations of seasonal means (summer, rainy, winter) and monthly means. Differences in seasonal and monthly means were determined using a generalized least square (GLS) method for repeated measures by R 3.0.0 (R Development Core Team, 2013) with nlme package (Pinheiro et al., 2013). Behavioral data were analyzed on
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
73
Fig. 1. Reverse-phase HPLC separation of immunoreactive androgen metabolites in feces of goral. Immunoreactivity in each fraction was measured with a testosterone EIA. Elution of the 3 H-testosterone reference tracer in HPLC fractions of extracted fecal samples is indicated by the arrow.
a seasonal basis only because of the low (or no) frequency of behaviors for some months. The structure of the covariance pattern for GLS was defined as a compound symmetry. A Bonferroni method was used for the multiple comparison adjustment. Relationships between weekly androgen and glucocorticoid metabolite concentrations and the association of each hormone to the environmental data (average temperature, rainfall, and day length) were analyzed using GLS method because of the correlated data. Pearson’s correlation coefficient analyses were used to determine the correlation in percent binding between serial dilutions of hormone standards and fecal extract dilutions in assay parallelism validation tests. For all statistical tests, the significance level was set at ˛ = 0.05. 3. Results Analysis of HPLC-separated male goral fecal eluates for immunoreactivity using the testosterone EIA revealed the presence of several androgen metabolites, one of which corresponded to radiolabeled testosterone (fractions 31–37; 25% of the immunoactivity) (Fig. 1). Three other more polar metabolite peaks occurred before the elution of testosterone at fractions 8–16 (33%), 17–23 (25%) and 24–28 (12%). Two comparatively smaller, less polar immunoreactive peaks occurred after testosterone at fractions 41–46 (4%) and 50–58 (7%).
A summary of male goral sexual behaviors across seasons is shown in Table 2. In general, males exhibited greater rates of tongue flick, approach and flehmen during the winter season (P < 0.05). Flehmen also was greater in the preceding rainy season (P < 0.05). During the study period, six of the eight male goral (62.5%) bred successfully, based on live births around 7 months later (7.07 ± 0.14 months) in July (n = 2 births), August (n = 1), October (n = 2) and December (n = 1). A number of copulations were observed for each female. Of the six that became pregnant, five copulated once during the conceptive cycle, whereas one bred in August before conceiving after one breeding in December. Two females did not conceive, although breeding was observed (3 and 1 mating each) in May and July, respectively. Overall mean fecal androgen and glucocorticoid metabolite concentrations across season during the study period are shown in Table 3. Average androgen concentrations were greatest in the rainy season and lowest in the summer, with intermediate concentrations in the winter (P < 0.05). Weekly and monthly mean androgen metabolite concentrations across the three seasons are shown in Fig. 2. Compared to the lesser concentrations observed in March, fecal androgens were greater in June to August. Concentrations in July also were greater than those in April and February (P < 0.05). There were positive correlations between fecal androgen metabolite concentrations
Table 2 Mean frequency (±SEM) of male goral sexual behaviors across seasons at Omkoi Wildlife Sanctuary, Chiang Mai, Thailand based on instantaneous scan sampling during 30-min observation periods. Behavior descriptions are based on Xie (2006). Season
Tongue flicka
Approachb
Smell and lickc
Flehmend
Mounte
Summerf Rainyf Winterf
1.32 ± 0.22ab 0.73 ± 0.27a 2.48 ± 0.51b
2.31 ± 0.25a 4.36 ± 2.58ab 4.57 ± 0.38b
1.10 ± 0.38a 1.36 ± 0.54a 2.36 ± 0.47a
0.42 ± 0.10a 1.27 ± 0.47b 1.05 ± 0.17b
0.31 ± 0.31a 0.73 ± 0.73a 1.21 ± 0.53a
Values differ among seasons, different superscript letters (a,b) indicate differences (P < 0.05). a Tongue flick – males flicks tongue between lips toward the female. b Approach – male walks directly toward the female. c Smell and lick – male approaches the female when she is laying down to sniff and lick, sometimes involving naso-genital contact. d Flehmen – male lifts head and curls the lips after sniffing female or her urine. e Mount – male rises on hind legs and rests forelegs and chest on the female. f Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
74
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Fig. 2. (a) Monthly mean (±SEM) androgen metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center. Values differ among seasons, different letters (a,b,c,d) indicate differences (P < 0.05). (b) Longitudinal mean (±SEM) androgen metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center.
and temperature (y = −12.88 + 11.19 × average temperature, P < 0.05) and day length (y = −134.49 + 31.75 × day length, P < 0.05) across the year. Mean glucocorticoid metabolite concentrations were less in the summer (P < 0.05), with no difference between rainy and winter seasons (Table 3), a pattern that is discernible in Fig. 3. Similar to androgens and compared to March, glucocorticoid concentrations were elevated in June to August and again in November to January (i.e., the rainy and winter seasons) (P < 0.05). In contrast to androgens, there were no correlations between glucocorticoid metabolite concentrations and mean temperature and day length. However, glucocorticoids were negatively correlated with rainfall (y = 21.57 − 0.24 × rainfall, P < 0.05). There also was a positive relationship between androgen and glucocorticoid metabolite concentrations (y = 64.82 + 8.82 × cortisol, P < 0.05) for the year. 4. Discussion This is the first study to characterize steroid hormone profiles in male goral, and confirmed the influence of seasonality on testicular and adrenal function. Overall concentrations of fecal androgen and glucocorticoid metabolites were greater in the rainy season and remained elevated throughout most of the winter before decreasing in the summer. The increase corresponded with successful copulations starting in July in the Omkoi males. Fecal androgen concentrations were positively correlated with temperature and day length, although glucocorticoids were not. Overall, results indicate that testicular and adrenal
steroidogenic activity in captive-held goral in Thailand follow a modest, but significant seasonal pattern, and at least for testicular activity may be initiated in part by subtle changes in photoperiod. A previous study validated the cortisol EIA for quantifying glucocorticoid metabolites in goral feces, including showing that native cortisol is excreted in appreciable amounts (17.5% of total immunoreactivity; Khonmee et al., unpublished). This study found that native testosterone also is excreted in goral feces (25%) in addition to other primarily more polar metabolites. Other species excrete native testosterone in feces in varying proportions [hyena (Crocuta crocuta), Dloniak et al., 2004); rhinoceros (Ceratotherium simum simum), Kretzschmar et al., 2004; bison (Bison bison bison),Mooring et al., 2004; chinchilla (Chinchilla lanigera), Busso et al., 2005], although not all do (felids, Brown et al., 1996; primates, Möhle et al., 2002). Using the EIA in the present study, the pattern of goral androgen production was similar to that of other seasonal ungulates (reindeer (Rangifer tarandus L.), Mossing and Damber, 1981; white-tailed deer (Odocoileus virginianus), Bubenik et al., 1987; wild and domestic sheep, Lincoln et al., 1990; Pelletier et al., 2003; impala (Aepyceros melampus), Brown et al., 1991; red deer (Cervus elaphus), Freudenberger et al., 1993; bison, Mooring et al., 2004), including several caprine species (Pérez and Mateos, 1995; Todini et al., 2007; Zarazaga et al., 2010; Polat et al., 2011), where concentrations increase preceding or in conjunction with rut or breeding activity. Chinese goral are reported to be distinctly seasonal with breeding occurring in the winter (October–January:
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
75
Fig. 3. (a) Monthly mean (±SEM) glucocorticoid metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center. Values differ among seasons, different letters (a,b,c,d) indicate differences (P < 0.05). (b) Longitudinal mean (±SEM) glucocorticoid metabolite concentrations in fecal samples collected from male goral housed at Omkoi Wildlife Breeding Center.
Kanbunjong, 1993; November–December: Lekagul and McNeely, 1988), a time frame that agrees with that for captive and wild Himalayan and red goral (Lovari and Apollonio, 1994; Xie, 2006; Grubb, 2005; Duckworth and MacKinnon, 2008). The closely related serow species (now in the genus Capricornis) also breeds primarily between November and January in situ (Duckworth et al., 2008). And sika deer (Cervus nippon), another Asian species, exhibit increased excreted gonadal steroids in both sexes during October–January (Yamauchi et al., 1997; Hamasaki et al., 2001). Thus, many if not most seasonal ungulates in Asia, including Thailand, breed toward the end of the year, during the cooler months of winter. At Omkoi however, androgen metabolites began to increase several months earlier, in July, and remained elevated throughout both rainy and winter season months. Females also displayed estrus earlier in the year based on observations of tail-up behaviors, which were accompanied by conceptive matings as early as 21 July. Thus, it appears that captive goral in Thailand, at least at Omkoi, breed earlier than wild counterparts, and exhibit an overall longer breeding season. This could be a captivity effect; some seasonal ungulates undergo changes in reproductive patterns, becoming less seasonal, non-seasonal or exhibiting extended breeding seasons ex situ (Skinner et al., 2002; Zerbe et al., 2012; Kaumanns et al., 2013). Reproductive seasonality is generally driven by two forces, alone or in combination: photoperiod and resource availability (Bronson, 1989; Kaumanns et al., 2013). There
were moderate differences across seasons in temperature and day length at Omkoi, about 3.5 ◦ C and 1.5 h from shortest to longest, respectively, and these were correlated, modestly though significantly, with fecal androgens. Thus, reproductive seasonality in goral may be regulated in part by photoperiod changes, a finding that has been well documented in other ungulates (see review, Zerbe et al., 2012). The Omkoi Wildlife Breeding Center is located inside a wildlife sanctuary where wild goral live, so the assumption was that captive and wild animals are exposed to similar environmental and photoperiodic conditions. However, there were elevation and likely climatic differences; wild goral prefer rocky crags at high elevations (1400–1929 m; Chaiyarat et al., 1999), whereas the elevation of the captive breeding center was much lower at only ∼500 m.
Table 3 Mean (±SEM) fecal androgen and glucocorticoid metabolite concentrations in male goral across seasons at Omkoi Wildlife Sanctuary, Chiang Mai, Thailand. Season
Androgen metabolites (ng/g)
Glucocorticoid metabolites (ng/g)
Summera Rainya Wintera
195.48 ± 8.23a 289.82 ± 9.18c 224.09 ± 11.97b
15.30 ± 0.48a 22.10 ± 0.72b 21.98 ± 0.98b
Values differ among seasons, different supercript letters (a,b,c) indicate differences (P < 0.05). a Summer (February 16–May 15), rainy (May 16–October 15) and winter (October 16–February 15).
76
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Another possibility for the early onset of breeding activity could be related to resources. Omkoi animals are fed natural forage, but also are provisioned with a pelleted concentrate daily. Thus, body condition remained relatively constant throughout the year compared to wild counterparts. Others have demonstrated the importance of good nutrition and body condition on enhancing reproductive function; for example, in both wild (Albon et al., 1986) and captive (Heydon et al., 1992; Carrion et al., 2008; GasparLópez et al., 2010) deer. So, males at Omkoi may attain a functional breeding state sooner than expected because of better overall body condition earlier in the season. Types of sexual behaviors of Chinese goral were similar to those reported for the Himalayan (Lovari and Apollonio, 1994) and red (Xie, 2006) goral, and included approach, smell and lick, tongue flick, and flehmen. At Omkoi, the frequency of behaviors was generally greater in the winter, and significantly for tongue flick and approach. Interestingly, this was after most of the females had conceived in this study. The flehmen response, however, was greater in both the rainy and winter seasons, when most of the conceptions occurred. Approach was the behavior most frequently observed at Omkoi, whereas in red goral smell and lick were most common (Xie, 2006). In Himalayan goral, the most common courtship behaviors were approach, following, mount and naso-nasal contact (Lovari and Apollonio, 1994). Both of these previous studies were conducted during the purported breeding season only (October for Lovari and Apollonio, 1994; September–November for Xie, 2006), however, so behaviors outside the breeding season were not documented. Still, considering these reports, it was surprising to find that males at Omkoi exhibited sexual behaviors throughout the year, as did females (e.g., tailup). However, greater androgen metabolite concentrations were observed during the months of active breeding, not unlike that reported for other ungulate species (Li et al., 2000, 2001; Imwalle et al., 2002; Mooring et al., 2004). Although there were no correlations between fecal glucocorticoid metabolite concentrations and temperature or day length, as with androgens, overall fecal glucocorticoid metabolites exhibited a clear seasonality, with concentrations being greater during the rainy and winter seasons as compared to the summer. Other ungulate species exhibit increased glucocorticoid production during winter [whitetailed deer (Odocoileus virginianus), Bubenik et al., 1983; Mccoy and Ditchkoff, 2012; pygmy goat (Capra hircus), Howland et al., 1985; mule deer (Odocoileus hemionus), Saltz and White, 1991] or late fall (bison, Mooring et al., 2006) seasons. Others, however, have no distinct seasonal glucocorticoid fluctuations, such as axis deer (Axis axis) (Bubenik and Brown, 1989), Eld’s deer (Cervus eldi thamin) (Monfort et al., 1993), pudu (Pudu pudu) (Reyes et al., 1997) and reindeer (Rangifer tarandus) (Bubenik et al., 1998). Differences in adrenal patterns may be species specific; however, it often is difficult to determine if increases in concentrations of glucocorticoids related to reproduction and/or androgen production are due to environmental or physiological effects when the two change in concert, as observed in goral. Although increased glucocorticoid concentrations during some types of stress can suppress testicular function and correlate negatively with
androgen secretion (baboon, Sapolsky, 1985; rat, Orr and Mann, 1992; guinea pig, Fenske, 1997; mice, Dong et al., 2004), co-production of androgens and glucocorticoids is observed during the normal breeding season in many ungulate species as noted above, including goral. 5. Conclusion This is the first study to assess male endocrine function in goral and to ascertain seasonal variation in testicular and adrenal steroidogenic function with greater activity in the rainy season and winter. Although wild goral breed primarily during the winter months, results of the present study indicate that captive males can be reproductively functional as early as July, and remain fertile through December. The function of enhanced adrenal activity during the rainy season and winter in male goral is not clear, but may be due to environmental conditions or breeding activity or both. Given that resources for goral at Omkoi are consistent throughout the year, reproduction may be regulated to a great extent by photoperiod. However, the early onset and extended breeding season at Omkoi suggests that maintaining consistently good body condition throughout the year may have a positive influence on initiation of spermatogenic function and reproductive behavior. Alternatively, being housed at a comparatively lower elevation compared to wild goral could have altered physiological responses to season. Knowledge of how endocrine function changes seasonally in goral can be used to enhance captive breeding, such as determining optimal times for semen collection and genome resource banking in conjunction with the use of ART procedures. Although semen analyses have yet to be been conducted, it appears that functional spermatozoa probably can be collected within 2–3 months after the start of the rainy season through the end of winter. Acknowledgments This study was supported by National Research Council of Thailand (NRCT) and the Graduate School of Chiang Mai University. The authors extend their gratitude to the staff of the Zoological Park Organization of Thailand, and Omkoi Wildlife Sanctuary for their assistance in sample collection. We are also grateful to Ms. Nicole Presley at the Smithsonian Conservation Biology Institute, and staff of the endocrinology lab at the Faculty of Veterinary Medicine, Chiang Mai University (Ms. Manisorn Tuantammarak, Ms. Patharanun Wongchai, and Mr. P allop Tankaew) for technical support. References Albon, S.D., Mitchell, B., Huby, B.J., Brown, D., 1986. Fertility in female red deer (Cervus elaphus): the effects of body composition, age and reproductive status. J. Zool. 209, 447–460. Bronson, F.H., 1989. Mammalian Reproductive Biology. University of Chicago Press, Chicago. Brown, J.L., Terio, K.A., Graham, L.H., 1996. Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids. Zoo Biol. 15, 425–434. Brown, J.L., Wasser, S.K., Wildt, D.E., Graham, L.H., 1994. Comparative aspects of steroid hormone metabolism and ovarian activity in felids, measured noninvasively in feces. Biol. Reprod. 51, 776–786.
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78 Brown, J.L., Wildt, D.E., Raath, J.R., de Vos, V., Janssen, D.L., Citino, S.B., Howard, J.G., Bush, M., 1991. Seasonal variation in pituitary-gonadal function in free-ranging impala (Aepyceros melampus). J. Reprod. Fertil. 93, 497–505. Bubenik, G.A., Brown, R.D., 1989. Seasonal levels of cortisol, triiodothyronine and thyroxine in male axis deer. Comp. Biochem. Physiol. A: Physiol. 92, 499–503. Bubenik, G.A., Bubenik, A.B., Schams, D., Leatherland, J.F., 1983. Circadian and circannual rhythms of LH, FSH, testosterone, prolactin, cortisol, T3 and T4 in plasma of mature, male white-tailed deer. Comp. Biochem. Physiol. A: Physiol. 76, 37–45. Bubenik, G.A., Pomerantz, D.K., Schams, D., Smith, P.S., 1987. The role of androstenedione and testosterone in the reproduction and antler growth of a male white-tailed deer. Acta Endocrinol. 114, 147–152. Bubenik, G.A., Schams, D., White, R.G., Rowell, J., Blake, J., Bartos, L., 1998. Seasonal levels of metabolic hormones and substrates in male and female reindeer (Rangifer tarandus). Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 120, 307–315. Busso, J.M., Ponzio, M.F., Dabbene, V., Cuneo, M.F., de Ruiz, R.D., 2005. Assessment of urine and fecal testosterone metabolite excretion in Chinchilla lanigera males. Anim. Reprod. Sci. 86, 339–351. Carrion, D., Garcıa, A.J., Gaspar-Lopez, E., Landete-Castillejos, T., Gallego, L., 2008. Development of body condition and its effect on calf birth weight and milk production. J. Exp. Zool. 309A, 1–10. Chaiyarat, R., Laohajinda, W., Kutintara, U., Nabhitabhata, J., 1999. Ecology of the goral (Naemorhedus goral) in Omkoi Wildlife Sanctuary Thailand. Nat. Hist. Bull. SIAM Soc. 47, 191–205. Conde, D.A., Flesness, N., Colchero, F., Jones, O.R., Scheuerlein, A., 2011. An emerging role of zoos to conserve biodiversity. Science 331, 1390–1391. Dloniak, S.M., French, J.A., Place, N.J., Weldele, M.L., Glickman, S.E., Holekamp, K.E., 2004. Non-invasive monitoring of fecal androgens in spotted hyenas (Crocuta crocuta). Gen. Comp. Endocrinol. 135, 51–61. Dong, Q., Salva, A., Sottas, C.M., Niu, E., Holmes, M., Hardy, M.P., 2004. Rapid glucocorticoid mediation of suppressed testosterone biosynthesis in male mice subjected to immobilization stress. J. Androl. 25, 973–981. Duckworth, J.W., Steinmetz, R., Chaiyarat, R., 2008. Naemorhedus griseus. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2 [WWW Document]. http://www.iucnredlist.org (accessed 23.4.13). Duckworth, J.W., MacKinnon, J., 2008. Naemorhedus caudatus. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2013.1 [WWW Document]. http://www.iucnredlist.org/details/14294/0 (accessed 8.8.13). Fenske, M., 1997. Role of cortisol in the ACTH-induced suppression of testicular steroidogenesis in guinea pigs. J. Endocrinol. 154, 407–414. Freudenberger, D.O., Wilson, P.R., Barry, T.N., Sun, Y.X., Purchas, R.W., Trigg, T.E., 1993. Effects of immunization against GnRH upon body growth, voluntary food intake and plasma hormone concentration in yearling red deer stags (Cervus elaphus). J. Agric. Sci. 121, 381–388. Gaspar-López, E., Landete-Castillejos, T., Estevez, J., Ceacero, F., Gallego, L., García, A., 2010. Biometrics, testosterone, cortisol and antler growth cycle in Iberian red deer stags (Cervus elaphus hispanicus). Reprod. Domest. Anim. 45, 243–249. Grubb, P., 2005. Order Artiodactyla. In: Wilson, D.E., Reeder, D.M. (Eds.), Mammal Species of the World: A Taxonomic and Geographic Reference. , third ed. The Johns Hopkins University Press, Baltimore, pp. 637–722. Hamasaki, S., Yamauchi, K., Ohki, T., Murakami, M., Takahara, Y., Takeuchi, Y., Mori, Y., 2001. Comparison of various reproductive status in sika deer (Cervus nippon) using fecal steroid analysis. J. Vet. Med. Sci. 63, 195–198. Heydon, M.J., Loudon, A.S.I., Milne, J.A., Brinklow, K.R., 1992. Influence of plane of nutrition on seasonal changes in food intake and reproduction in grazing red deer. In: Brown, R.D. (Ed.), Antler Development in Cervidae. Caesar Kleberg Wildlife Research Institute, Kingsville, pp. 279–284. Howland, B.E., Sanford, L.M., Palmer, W.M., 1985. Changes in serum levels of LH FSH, prolactin, testosterone, and cortisol associated with season and mating in male pygmy goats. J. Androl. 6, 89–96. Huber, S., Palme, R., Arnold, W., 2003. Effects of season, sex, and sample collection on concentrations of fecal cortisol metabolites in red deer (Cervus elaphus). Gen. Comp. Endocrinol. 130, 48–54. Imwalle, D.B., Daxenberger, A., Schillo, K.K., 2002. Effects of melengestrol acetate on reproductive behavior and concentrations of LH andtestosterone in bulls. J. Anim. Sci. 80, 1059–1067. Kanbunjong, S., 1993. Behavior of Goral (Naemorhaedus griseus) in Captivity. Chiang Mai University, Chiang Mai. Kretzschmar, P., Gansloßer, U., Dehnhard, M., 2004. Relationship between androgens, environmental factors and reproductive behavior in male
77
white rhinoceros (Ceratotherium simum simum). Horm. Behav. 45, 1–9. Lekagul, B., McNeely, J.A., 1988. Mammals of Thailand. Darnsutha Press, Bangkok. Li, C., Jiang, Z., Fang, J., Jiang, G., Ding, Y., Shen, H., Xu, A., 2000. Relationship between reproductive behavior and fecal steroid in milu (Elaphurus davidianus). Acta Theriol. Sin. 20, 88–100. Li, C., Jiang, Z., Jiang, G., Fang, J., 2001. Seasonal changes of reproductive behavior and fecal steroid concentrations in Père David’s deer. Horm. Behav. 40, 518–525. Lincoln, G.A., Lincoln, C.E., McNeilly, A.S., 1990. Seasonal cycles in the blood plasma concentration of FSH, inhibin and testosterone, and testicular size in rams of wild, feral and domesticated breeds of sheep. J. Reprod. Fertil. 88, 623–633. Lovari, S., Apollonio, M., 1994. On the rutting behaviour of the Himalayan goral Nemorhaedus goral (Hardwicke, 1825). J. Ethol. 12, 25–34. Mccoy, J.C., Ditchkoff, S.S., 2012. Patterns of fecal hormones in a fenced population of white-tailed deer. Wildl. Soc. Bull. 36, 641–646. Millspaugh, J.J., Washburn, B.E., 2004. Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation. Gen. Comp. Endocrinol. 138, 189–199. Möhle, U., Heistermann, M., Palme, R., Hodges, J.K., 2002. Characterization of urinary and fecal metabolites of testosterone and their measurement for assessing gonadal endocrine function in male nonhuman primates. Gen. Comp. Endocrinol. 129, 135–145. Monfort, S.L., Brown, J.L., Wildt, D.E., 1993. Episodic and seasonal rhythms of cortisol secretion in male Eld’s deer (Cervus eldi thamin). J. Endocrinol. 138, 41–49. Mooring, M.S., Patton, M.L., Lance, V.A., Hall, B.M., Schaad, E.W., Fetter, G.A., Fortin, S.S., McPeak, K.M., 2006. Glucocorticoids of bison bulls in relation to social status. Horm. Behav. 49, 369–375. Mooring, M.S., Patton, M.L., Lance, V.A., Hall, B.M., Schaad, E.W., Fortin, S.S., Jella, J.E., McPeak, K.M., 2004. Fecal androgens of bison bulls during the rut. Horm. Behav. 46, 392–398. Mossing, T., Damber, J.E., 1981. Rutting behavior and androgen variation in reindeer (Rangifer tarandus L.). J. Chem. Ecol. 7, 377–389. Orr, T.E., Mann, D.R., 1992. Role of glucocorticoids in the stress-induced suppression of testicular steroidogenesis in adult male rats. Horm. Behav. 26, 350–363. Patton, M.L., Aubrey, L., Edwards, M., Rieches, R., Zuba, J., Lance, V.A., 2000. Successful contraception in a herd of Chinese goral (Nemorhaedus goral arnouxianus) with melengestrol acetate. J. Zoo Wildl. Med. 31, 228–230. Pelletier, F., Bauman, J., Festa-Bianchet, M., 2003. Fecal testosterone in bighorn sheep (Ovis canadensis): behavioural and endocrine correlates. Can. J. Zool. 81, 1678–1684. Pérez, B., Mateos, E., 1995. Seasonal variations in plasma testosterone ˜ bucks. Small Rumin. Res. 15, 155–162. levels in Verata and Malaguena Pinheiro, J., Bates, D., DebRoy, S., Sarkar, D., the R Development Core Team, 2013. nlme: Linear and Nonlinear Mixed Effects Models. In: R Package Version 3.1-110. R Development Core Team, 2013. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3-900051-07-0 [WWW Document] http://www.R-project.org/ (accessed 1.7.13). Rabinowitz, A., Khaing, S.T., 1998. Status of selected mammal species in North Myanmar. Oryx 32, 201–208. Reyes, E., Bubenik, G.A., Lobos, A., Schams, D., Bartos, L., 1997. Seasonal levels of cortisol. IGF-1 and triiodothyronine in adultmale pudu (Pudu pudu). Fol. Zool. 46, 109–116. Rudert, S., Brown, J., Gansloßer, l., Möbius, U., Songsasen, G.N., 2011. Activity pattern, reproductive behaviors and gonadal hormones in the raccoon dog (Nyctereutes procyonoides). Zoo Biol. 30, 134–148. Saltz, D., White, G.C., 1991. Urinary cortisol and urea nitrogen responses to winter stress in mule deer. J. Wildl. Manage. 55, 1. Sapolsky, R.M., 1985. Stress-induced suppression of testicular function in the wild baboon: role of glucocorticoids. Endocrinology 116, 2273–2278. Skinner, J.D., Moss, D.G., Skinner, D.C., 2002. Inherent seasonality in the breeding seasons of African mammals: evidence from captive breeding. Trans. R. Soc. S. Afr. 57, 25–33. Thai Meteorological Department [WWW Document], 2013. http://www.tmd.go.th/en/ (accessed 24.4.13). Todini, L., Malfatti, A., Terzano, G.M., Borghese, A., Pizzillo, M., Debenedetti, A., 2007. Seasonality of plasma testosterone in males of four Mediterranean goat breeds and in three different climatic conditions. Theriogenology 67, 627–631.
78
J. Khonmee et al. / Animal Reproduction Science 146 (2014) 70–78
Xie, Y., 2006. Primary observations on rutting behavior of the captive red goral. Zoo Biol. 25, 117–123. Yamauchi, K., Hamasaki, S., Takeuchi, Y., Mori, Y., 1997. Assessment of reproductive status of sika deer by fecal steroid analysis. J. Reprod. Dev. 43, 221–226. Young, K.M., Brown, J.L., Goodrowe, K.L., 2001. Characterization of reproductive cycles and adrenal activity in the black-footed ferret (Mustela nigripes) by fecal hormone analysis. Zoo Biol. 20, 517–536.
Zarazaga, L.A., Gatica, M.C., Celi, I., Guzmán, J.L., Malpaux, B., 2010. Effect of artificial long days and/or melatonin treatment on the sexual activity of Mediterranean bucks. Small Rumin. Res. 93, 110–118. Zerbe, P., Clauss, M., Codron, D., Bingaman Lackey, L., Rensch, E., Streich, J.W., Hatt, J.-M., Müller, D.W.H., 2012. Reproductive seasonality in captive wild ruminants: implications for biogeographical adaptation, photoperiodic control, and life history. Biol. Rev. Camb. Philos. Soc. 87, 965–990.