Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus)

Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in... Volume 5 � 2017 10.1093/conphys/cox001 Research article Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus) 1 2 3 1 Jaruwan Khonmee , Suvichai Rojanasthien , Chatchote Thitaram , Jureerat Sumretprasong , 4 5 6, Anurut Aunsusin , Chawin Chaisongkram and Nucharin Songsasen Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Department of Companion Animal and Wildlife Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Chiang Mai Night Safari, Hang Dong, Chiang Mai 50230, Thailand Chiang Mai Zoo, Chiang Mai 50200, Thailand Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA *Corresponding author: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA. Tel: +1 540 635 0030. Email: songsasenn@si.edu .............................................................................................................................................................. To date, there is no information on reproductive endocrinology of dholes (Cuon alpinus). The objectives of the present study were as follows: (i) to characterize longitudinal profiles of gonadal steroids; and (ii) to examine the relationship between gonadal hormones and sexual behaviours in dholes. Three breeding pairs and two bachelor males were included in the study. Among these, four animals (2 males and 2 females; 4 years old) were imported from The Netherlands to Thailand 3 months before the study onset; the remaining individuals (3 males and 1 female; 5–7 years old) were native born. Faecal samples were collected 3–7 days/week for 12 months, extracted and assessed for gonadal hormone metabolites using a validated enzyme immunoassay. Observations of behaviour were conducted in 30 min sessions, 3–5 days/week. For the three breeding males, testosterone was elevated (P < 0.05) from October to January in the two imported males, whereas the concentration of steroid metabolites was high from April to June and from September to November in the native male. However, there was no clear seasonal pattern of reproductive hormone in the bachelor group. Oestrogen metabolite level of imported females was elevated for 9–12 days in January, followed by a rise in progestagen concentration. For native females, oestrogen metabolites were above the basal values in April and September, each of which was followed by a rise in progestagen concentration that remained elevated for 77 and 112 days, respectively. Sexual behaviours, including solicitation, mount- ing and copulations, were observed during the oestrogen peak in all females. Our findings indicate that reproductive sea- sonality of dholes may depend on the animals’ origin and social group. Key words: Dhole, gonadal steroids, non-invasive hormone monitoring, sexual behaviour Editor: Steven Cooke Received 10 October 2016; Revised 26 December 2016; Editorial Decision 2 January 2017; accepted 9 January 2017 Cite as: Khonmee J, Rojanasthien S, Thitaram C, Sumretprasong J, Aunsusin A, Chaisongkram C, Songsasen N (2017) Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus). Conserv Physiol 5(1): cox001; doi:10.1093/ conphys/cox001. .............................................................................................................................................................. .............................................................................................................................................................. © The Author 2017. Published by Oxford University Press and the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Introduction Materials and methods The dhole or Asiatic wild dog (Cuon alpinus) is a medium- Animals and collection of samples sized, social canid. The species currently inhabits a fragmen- A total of five males and three females maintained in two zoos ted range in southern China, India, Myanmar, Thailand, in Chiang Mai, Thailand (18° 47′ 25″N, 98° 58′ 54″E) were Laos, Vietnam, Malaysia and Indonesia (Durbin et al., included in the study. The three breeding pairs were housed at 2004). Dholes are listed as ‘endangered’ by the International the Chiang Mai Night Safari, two (4 years old) of which were Union for Conservation of Nature (IUCN, 2016), mainly imported from The Netherlands 3 months before the study because of threats associated with habitat fragmentation, onset, and were kept off exhibit in a 3 m × 4 m (width × hunting, prey depletion, competition with other large carni- length) outdoor enclosure. During the study period, the two vores (i.e. tigers and leopards) and diseases transmitted by imported females gave birth; one had one pup that died soon feral/domestic dogs (Durbin et al., 2004). It has been esti- after birth at the end of March and another had a litter of mated that there are <2500 mature individuals living the four pups at the beginning of April, one of which survived wild (IUCN, 2016). The dhole is secretive and lives in highly (hand rearing). The remaining pair (7 years old), born in social, close-knit packs of three to 20 individuals, with a Thailand were maintained on exhibit in a 5 m × 4 m (width × structure of rigid dominance hierarchies (Durbin et al., length) outdoor enclosure. The two bachelor males (5 years 2004). Dholes hunt co-operatively and maintain communica- old) were born in Thailand and kept on exhibit 3 m × 4m tion with pack members by whistling as they move through (width × length) at Chiang Mai Zoo. All animals received dense forests (Fox, 2004). natural light and were fed fresh chicken once daily. Fresh fae- cal samples (~30 g) were collected 3–7 days/week for males To date, there is limited information on dhole reproduc- and 5–7 days/week for females for 12 months. All samples tion. Behavioural data gleaned from a 4 year study in India were stored at −20°C until hormone extraction and analysis. suggested that dholes are seasonal breeders (Paulraj et al., All animal procedures were approved by the National Zoo’s 1992). However, the time of breeding season varies among Institutional Animal Care and Use Committee. regions. Paulraj et al. (1992) reported that breeding occurs between August and December in India. However, a study Faecal extraction conducted in the same country revealed that mating occurs between November and April, with peak activity observed All chemicals were obtained from Sigma Chemical Company during December and January (Durbin et al., 2004). Finally, (St Louis, MO, USA), unless otherwise stated. Frozen faecal dholes are thought to mate mainly during January and May samples were thawed at room temperature, and 0.5 g of each in East Java (Durbin et al., 2004), and those housed in a zoo well-mixed wet sample was placed in a glass tube containing in Germany breed in January (Maisch, 2010). The oestrous 90% ethanol, vigorously shaken in a Multi Pulse vortexer period lasts 14–39 days, and sexual behaviours, including (Glas-Col, Terre Haute, IN, USA) for 30 min, and centri- solicitation, copulatory tie and back-to-back posture have fuged at 1300 g for 20 min. The supernatants were stored at been observed during this period, as in other canids; the −20°C until further analysis. Extraction efficiencies, deter- copulation period ranged from 1 to 9 days (Durbin et al., mined by addition of 2500 d.p.m. H-steroid before extrac- 2004). Based on behavioural observations, dholes exhibited tion were 96.2% [coefficient of variance (CV) <10%] for seasonal polyoestrus with a period of 4–6 weeks between testosterone (male samples) and 88.9% (CV <10%) for pro- cycles (Durbin et al., 2004), which is different from other gesterone (female samples). wild canids, such as the maned wolf (Chrysocyon bra- chyurus; Velloso et al., 1998; Songsasen et al., 2006), Enzyme immunoassay African wild dog (Lycaon pictus; Monfort et al., 1997) and red wolf (Canis rufus; Walker et al., 2002). The gestation Testosterone, oestrogen and progestagen metabolites were period is ~9 weeks (Durbin et al., 2004; Fox, 2004), and lit- quantified by enzyme immunoassay. Antibodies for testoster- ter size ranges from four to 12 pups (Durbin et al., 2004). one (polyclonal testosterone R156/7; 1:8500 dilution), oestro- gen (polyclonal estrone conjugate ECR522; 1:20 000 dilution) To date, there is no information on reproductive endocrin- and progesterone (monoclonal pregnane CL425; 1:10 000 ology of the dhole. Therefore, the main goal of the present dilution) were obtained from the University of California, study was to characterise longitudinal gonadal steroid profiles Davis, CA, USA. Cross-reactivities of R156/7 and ECR522 using a non-invasive endocrine monitoring technique devel- have previously been reported by Munro et al. (1991), oped for domestic and wildlife species (Schwarzenberger et al., whereas that of CL425 was reported by Graham et al. (2001). 1996; Brown, 1995, 2011). The specific objectives were as fol- lows: (i) to determine the influence of seasonality on gonadal Serial dilutions of pooled faecal extracts produced dis- hormone excretion; and (ii) to examine the relationship placement curves parallel to those of the appropriate stan- between reproductive hormones and sexual behaviours in dards. Recovery of added standard to pooled faecal extracts females. demonstrated significant (P < 0.05) recovery (testosterone, .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. y = 0.77x + 39.47, R = 0.997; oestrogen, y = 1.16x + Oestrogen and progestagen metabolites 0.28, R = 0.998; and progestagen, y = 0.79x + 2.30, Longitudinal profiles of oestrogen and progestagen metabo- R = 0.998). Inter-assay CVs were <15% and intra-assay lites were aligned to the day of the oestrogen peak (day 0). CVs <10%. Assay sensitivities were 2.3, 1.95 and 0.78 pg/ Day 0 was identified as the first day that oestrogen rose well for testosterone, oestrogen and progestagen analyses, above baseline concentration by 2 SD (Songsasen et al., respectively. 2006). Baseline values for each individual were calculated by an iterative process, whereby high values (exceeding the High-performance liquid chromatography mean plus 1.5 SD) were excluded. Each time the average was recalculated, and the elimination process repeated until no The quantities and relative proportions of immunoactive values exceeded the mean plus 1.5 SD. Data were reported as testosterone, estrogen and progestagen metabolites in dhole means ± SEM. Longitudinal steroid metabolite profiles were faecal extracts were determined using reverse-phase high-per- divided into three reproductive stages using modified criteria formance liquid chromatography (HPLC; Microsorb C-18 of those described by Velloso et al. (1998) and Walker et al. Column, Rainen Inc., Woburn, MA, USA). For each sex, five (2002), as follows: (i) anoestrus (days −30 to −11 before the faecal extracts were combined, air dried, resuspended in 1 ml oestrogen peak); (ii) pro-oestrus (days −10 to −1); (iii) oestrus methanol, dried under air and stored at −20°C until analysis. (days 0–6); and (iv) dioestrus (days 7–93 or until the end of Extract pools were reconstituted with 0.5 ml phosphate- faecal collection in the two imported females). Differences in buffered saline and filtered through a C-18 spice cartridge faecal steroid concentration among different stages of repro- (VWR, West Chester, PA, USA), eluted with 5 ml methanol, ductive cycles (females) within the same individuals were air dried and spiked with known radio-labelled steroids ( H- 3 3 3 determined by ANOVA followed by the Holm–Sidak meth- testosterone, H-estradiol-17β, H-estrone, H-estrone-sulfate od for multiple comparison. Differences were considered sig- and H-progesterone, ~2500 d.p.m. each). Subsequently, the nificant when the P-value was <0.05. samples were dried down and resuspended in 300 µl metha- nol. Testosterone metabolites were separated using a gradient of 45% methanol over 90 min (1 ml/fraction; 1 ml/min flow Results rate). Oestrogen metabolites were separated using a 20–80% methanol gradient over 80 min, and progestagen metabolites High-performance liquid chromatography were separated using 20–30–50–100% acetonitrile gradients over 15, 45 and 60 min, respectively. Co-elution profiles of the Evaluation of faecal extracts by HPLC revealed the presence respective radio-labelled steroids in each HPLC run were deter- of several testosterone metabolites, one (10%) of which mined by adding 100 µlofeachHPLC fractionto 3ml of exhibited similar retention time to testosterone (fractions scintillation fluid (Ultima Gold; Packard, Meriden, CT, USA) 30–35; Fig. 1). The HPLC analysis of oestrogen metabolites and counted in a dual-label channel β scintillation counter demonstrated three immunoreactive peaks (fractions 15–20, (Beckman, Fullerton, CA, USA). Each of the remaining HPLC 57–61 and 60–63), which co-eluted with estrone-3-sulfate, fractions was then air dried, reconstituted in 0.2 ml assay buffer estrone and estradiol (Fig. 2). Progestagen immunoreactivity and quantified for immunoactivity by enzyme immunoassay. was associated with a single peak (fractions 63–68), which corresponded to the radio-labelled progesterone (Fig. 3). Behavioural observations Steroid metabolite concentrations of male Quantitative behavioural data were collected 3–5 days/week in all breeding females during 30 min observation sessions con- dholes ducted in the morning with instantaneous scan sampling Mean ± SEM testosterone metabolites of imported (breed- (Martin and Bateson, 2007). During the observation period, all ing), native (breeding) and native (bachelor) males are shown sexual behaviours, including solicitation, mounting and copu- in Fig. 4A–C. For breeding males, testosterone concentra- lation, were recorded using an ethogram adapted from that tions varied among months. However, there were individual developed for the maned wolf (Rodden et al., 1996). The differences in the time during which high testosterone con- behavioural data were then superimposed on endocrine profiles. centrations were observed. Specifically, testosterone metabol- ite concentrations of the two imported dholes (males A and Hormonal profiles and data analysis B) were at low levels during February–September. Steroid concentrations significantly increased in October and remained Testosterone elevated (P < 0.05) until January (Fig. 4A). Mean testosterone metabolites were calculated on a monthly basis and compared across months within each male to For the native male that was paired with a female (male C), determine the influence of seasonality on hormone produc- testosterone metabolite concentrations were low in February, tion using Kruskal–Wallis one-way analysis of variance slightly elevated in April–June, significantly declined in July (ANOVA) followed by Dunn’s method (SigmaStat 3.0; SPSS and August and then substantially increased in September and Inc., Chicago, IL, USA). Significance was set at 95%. remained at high concentrations until November (P < 0.05; .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Immunoactivity (ng/ml) Immunoactivity (ng/ml) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Testosterone Radioactivity 16000 3500 Immunoactivity 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 HPLC fractions Figure 1: Co-chromatographic high-performance liquid chromatography separation of faecal testosterone metabolites of male dholes. Radioactivity Estrone Estradiol 9000 40 Immunoactivity Estrone-3- sulfate 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 HPLC fractions Figure 2: Co-chromatographic high-performance liquid chromatography separation of faecal oestrogen metabolites of female dholes. Fig. 4B). Seasonal variations in testosterone concentrations during oestrus than in pro-oestrus, dioestrus and anoestrus. were not apparent in the two bachelor males (Fig. 4C). Progestagen metabolite concentrations were at the baseline level during pro-oestrus, slightly increased during oestrus, sig- nificantly elevated during dioestrus (P < 0.05) and returned to Steroid metabolite concentrations and the baseline levels during the anoestrous period. reproductive behaviours of female dholes In all females, the increase in oestrogen coincided with vul- Faecal oestrogen and progestagen concentrations during val swelling and sexual behaviours, including solicitation, various stages of the reproductive cycle are shown in Fig. 5. mounting and copulations (Fig. 6A–C). However, there were Regardless of the pregnancy outcome, all females exhibited a marked differences in reproductive season between imported similar endocrine pattern. Specifically, mean oestrogen metab- and native females. The oestrogen peak of the imported females olite concentrations in all females were higher (P < 0.05) was observed in January, followed by a rise in progestagen .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Radioactivity (cpm/ml) Radioactivity (cpm/ml) Immunoactivity (ng/ml) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. Progesterone Radioactivity Immunoactivity 7000 4000 0 0 1 112131415161718191 101 111 HPLC fractions Figure 3: Co-chromatographic high-performance liquid chromatography separation of faecal progestagen metabolites of female dholes. concentrations, which remained elevated until the beginning of that reported in the raccoon dog (Nyctereutes procyonoides; April (Fig. 6A and B). In the native pair, copulations were Rudert et al., 2011). Given that the majority of metabolites observed in April and September, but pups were not produced. were not associated with testosterone, this assay could not Two oestrogen peaks, 149 days apart, were observed in the be used as an exclusive index for this steroid hormone. native female (in April and September); each was followed by Nevertheless, variations in testosterone metabolite concentra- increased progestagen metabolite concentrations, which tions among seasons with high hormone concentrations remained elevated for 77 and 114 days, respectively (Fig. 6C). observed during breeding periods in paired individuals indi- cated that this assay can be used to assess gonadal functions in male dholes. Unlike the African wild dog (Monfort et al., 1997), red wolf (Walker et al., 2002) and maned wolf Discussion (Velloso et al., 1998), which excrete mostly estradiol and Non-invasive faecal hormone metabolite monitoring has estrone, dhole samples contained a significant amount of proved to be a valuable tool in advancing the understanding estrone-3-sulfate (20%) in addition to the two metabolites of reproductive functions and enhancing ex situ management found in other canids. The immunoreactive progestagen of various wildlife species, including carnivores (Brown et al., peak of dhole samples was co-eluted with progesterone, 1995). The present study characterized gonadal hormone pro- which also differed from the findings in the African wild dog files in male and female dholes and investigated the influence (Monfort et al., 1997), red wolf (Walker et al., 2002) and of seasonality on the steroid excretion patterns in this species. maned wolf (Velloso et al., 1998). In these three canids, pro- Our data demonstrated that dholes are seasonal breeders, with gestagen metabolites excreted in faecal samples were less gonadal hormone concentrations that varied among months, polar than progesterone. Given that increases in both oestro- especially in breeding pairs. Specifically, in imported males, tes- gen and progestagen concentrations coincided with reproduct- tosterone metabolites were elevated from October to January ive behaviours observed in the three pairs, these assays can be for the males, whereas steroid metabolites in the breeding used to evaluate reproductive functions in female dholes. native male were at a high level during April–June and To date, information regarding dhole reproductive biology September–November. The seasonal pattern of testosterone has been generated exclusively from behavioural observation excretion corresponded to sexual behaviours and increased of captive and wild individuals (Durbin et al., 2004; Maisch, gonadal function in the females. Our findings also suggested 2010). It has been suggested that female dholes exhibit sea- that intrinsic (e.g. genetic) factors, rather than environmental sonal polyoestrus with 4–6 weeks between the end of the first conditions, may influence the time during which reproduction and beginning of the next cycle (Durbin et al., 2004). In the occurs. Finally, our data suggested that the dhole is a polyoes- present study, we observed that the non-pregnant female trous canid, a characteristic that is unique among species (female C) exhibited two estrous cycles during the study peri- within the Canidae family. od, one from April to June and one from September to In the present study, it was found that only a small pro- December, whereas the two pregnant females cycled only once portion (10%) of androgen metabolites were excreted as tes- a year. Although our observation was based on one female, it tosterone in dhole faecal samples. This finding is similar to appeared that our endocrine data confirmed behavioural .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Radioactivity (cpm/ml) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Imported (breeding) male A Imported (breeding) male B ** ## ** ## ** ** ## 16000 b ab ab ab b ab ab a a Feb Mar April May June July Aug Sep Oct Nov Dec Jan Feb Native (bachelor) male D Native (bachelor) male E ** ** Figure 4: Mean ± SEM testosterone metabolite concentrations of two imported males housed in breeding pairs (A), a native male housed in a **, ##, ++ a,b breeding pair (B) and two native males housed as a bachelor group (C). Symbols or superscript letters indicate significant differences among months within the same male. P < 0.05. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Faecal testosterone metabolites Faecal testosterone metabolites Faecal testosterone metabolites (ng/g wet faeces) (ng/g wet faeces) (ng/g wet faeces) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. which was consistent with the oestrous period observed in Imported (breeding) female A Oestrogen the females. Given that the two imported pairs gave birth 10000 Progestagen in March and April, we estimated that breeding occurred in January, during the first reproductive season after they were transported to Thailand, and this pattern remained the same in the following year. This breeding period (January) differed from that observed in the native pair and wild counterparts living in Thailand (April and September). Specifically, the native pair exhibited breeding behaviours in April and ab ab a bc September, which coincided with increased gonadal metabol- b c b 0 ite concentrations in both male and female. A camera-trap Pro-oestrus Oestrus Dioestrus Anoestrus study conducted in eastern Thailand documented a photo- graph of two juveniles (~6 months old) in May, indicating Imported (breeding) female B that breeding probably occurred in September or October in Oestrogen wild individuals (Jenks et al.,2012). Furthermore, a lactating Progestagen female was captured in a Thai protected area in mid- 8000 February, suggesting that this individual probably bred in late October to early November (N.S., personal observation). Thus, it appears that the reproductive season of imported dholes in the present study did not shift to the same period as that of native animals, even after being in Thailand for 1 year. ab This is in contrast to observations in the maned wolf. The ab b b b maned wolf breeding season differs by 6 months between the Northern and Southern hemispheres, with the peak breeding Pro-oestrus Oestrus Dioestrus Anoestrus occurring in October (autumn) and April (autumn) in North 12000 and South America, respectively (Maia and Gouveia, 2002). Oestrogen Native (breeding) female C Analysis of faecal testosterone concentrations of a male maned a Progestagen wolf transported from Brazil to the USA in June (i.e. the end of breeding season in Brazil) showed that this individual adapted 8000 ab to the reproductive season of North America shortly after being placed in the new environment, as steroid concentrations remained elevated until the following January (i.e. the end of the breeding season in the USA; N.S., unpublished data). Thus, bc 2000 it appears that reproductive seasonality in the maned wolf is ab b ab regulated by environmental factors (photoperiod). The influence of photoperiod on reproduction has also been observed in the Pro-oestrus Oestrus Dioestrus Anoestrus red wolf (Canis rufus; Walker et al., 2002), in which variation in faecal androgen metabolite concentrations is linked to chan- Figure 5: Mean ± SEM concentrations of ovarian steroid metabolites during pro-oestrus (days −10 to −1 of the oestrogen peak), oestrus ging day length; steroid concentration increases in late autumn (days 0–6), dioestrus (days 7–93) and anoestrus (days −30 to −11) of (October) and peaks in winter (February). Given that imported the two imported females (A and B) and the native female (C). dholes maintained the same reproductive seasonal pattern abc Different superscript letters indicate significant differences among they had exhibited in Europe even after living in Thailand for reproductive cycles within the same individual for each steroid 1 year, we suspect that the reproductive season of this species hormone (P < 0.05). may be regulated by other factors, rather than the environment. Based on morphology, geographical locations and genetic observations of others (Durbin et al.,2004), suggesting that information, dholes are classified into 11 subspecies (Durbin dholes are unique amongst wild canids, which are seasonally et al., 2004; Iyengar et al., 2005). Recently, analysis of mito- monoestrous (Asa and Valdespino, 1998). Future studies that chondrial DNA of imported and native individuals included include multiple females are required to confirm this unique in the present study revealed that these two animal groups reproductive characteristic of the dhole. differed in the haplotype of mitochondrial control region Another interesting aspect is that there appeared to be dif- (J. Kayman, personal communication). Specifically, the for- ferences in reproductive season between imported and native mer was classified as having haplotype R, the type found individuals, at least during the study period (1 year). In the mostly in captive dholes housed in European zoos. However, imported males (males A and B), testosterone metabolites all native individuals were of haplotype U, a novel haplotype reached peak concentrations between October and January, that has not been previously described (Iyengar et al., 2005). .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Gonadal hormone metabolites Gonadal hormone metabolites Gonadal hormone metabolities (ng/g wet faeces) (ng/g wet faeces) (ng/g wet faeces) Faecal Progestagen (ng/g wet faeces) Faecal progestagen (ng/g wet faeces) Faecal Progestagen (ng/g wet faeces) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. 3500 39000 Oestrogen Progestagen Swollen vulva 2500 Elevated Testosterone Birth 0 0 Date Oestrogen 3500 39000 Progestagen Swollen vulva Elevated Testosterone Birth 0 0 Date Oestrogen 3500 Progestagen 39000 Mounting Copulations Elevated Testosterone 0 0 Date Figure 6: Longitudinal profile of gonadal steroids of the two imported females (A and B) and the native female (C) overlaid with reproductive events/behaviours and the interval during which elevated testosterone metabolites were observed in the respective male. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Faecal Oestrogen (ng/g wet faeces) Faecal Oestrogen (ng/g wet faeces) Faecal Oestrogen metabolites (ng/g wet faeces) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. Asher GW, O’Neill KT, Scott LC, Mockett BG, Fisher MW (2000) Genetic Thus, it is possible that the difference in seasonal cyclicity influence on reproduction in female red deer (Cervus elaphus): sea- between imported and native dholes may be, in part, asso- sonal luteal cyclicity. Anim Reprod Sci 28: 43–59. ciated with genetic divergence. The influence of genetic fac- tors on reproductive cyclicity has been previously shown in Brown JL (2011) Female reproductive cycle of wild female felids. Anim the red deer (Cervus elaphus; Asher et al., 2000). Reprod Sci 124: 155–162. By combining behavioural observation and endocrine moni- Brown JL, Wemmer CM, Lehnhardt J (1995) Urinary cortisol analysis toring, we have demonstrated that the dhole oestrous period for monitoring adrenal activity in elephants. Zoo Biol 14: 533–542. ranges from 9 to 13 days, which is similar to that reported in Concannon PW, Hansel W, McEntree K (1977) Changes in LH, proges- other canids, including the grey wolf (Canis lupus; Seal et al., terone and sexual behaviour associated with preovulatory luteini- 1987), fennec fox (Vulpes zerda; Valdespino et al.,2002), red zation in the bitch. Biol Reprod 17: 604–613. wolf (Walker et al.,2002), maned wolf (Songsasen et al.,2006) and domestic dog (Edqvist et al.,1975; Concannon et al., Durbin LS, Venkataraman A, Hedges S, Duckworth W (2004) Dhole 1977; Wildt et al.,1979). Behavioural studies conducted on (Cuon alpinus). In C Sillero-Zubiri, M Hoffman, DW Macdonald, eds, captive dholes reported that the oestrous period is 3–4 weeks, Canids: Foxes, Wolves, Jackals and Dogs. Gland, Switzerland: IUCN, and the copulation period ranges from 1 to 9 days (Paulraj pp 201–218. et al.,1992; Maisch, 2010). Courtship behaviours of dhole observed in the present study were similar to those reported in Edqvist LE, Jaohansson EDB, Kasström H, Olsson SE, Richkind M (1975) the previous study (Paulraj et al.,1992); these include mount- Blood plasma levels of progesterone and oestradiol in the dog ing, copulatory tie and back-to-back copulatory posture. In the during the oestrous cycle and pregnancy. Acta Endocrinol 78: present study, two oestrogen peaks 149 days apart were 554–564. observed in the native female; each was followed by increased Fox MW (2004) The Whistling Hunters: Field Studies of the Asiatic Wild progestagen metabolite concentrations that remained elevated Dog (Cuon alpinus). Albany: State University of New York Press. for 77 and 114 days, respectively. Given that neither reproduct- ive cycle resulted in pregnancy, the discrepancy in the length of Graham LH, Schwarzenberger F, Mostl E, Galama W, Savage A (2001) the dioestrous period remains to be elucidated. A versatile enzyme immunoassay for the determination of proges- tagens in feces and serum. Zoo Biol 20: 227–236. In summary, by using non-invasive hormone monitoring, IUCN (2016). The IUCN Red List of Threatened Species. Version 2016-3. we have established the first endocrine database characteris- www.iucn4redlist.org. ing the reproductive cycle of the endangered dhole. Despite the small sample size, longitudinal faecal steroid monitoring Iyengar A, Babu VN, Hedges SA, Venkataraman B, Maclean N, Morin through enzyme immunoassay assessments has revealed sev- PA (2005) Phylogeography, genetic structure, and diversity in the eral reproductive characteristics of this understudied canid, dhole (Cuon alpinus). Mol Ecol 14: 2281–2297. including the evidence of the following: (i) seasonal polyoes- trus in the females; and (ii) the potential of genetic impact on Jenks KE, Songsasen N, Leimgruber P (2012) Camera trap records of timing of reproductive seasonality. Future studies that dholes in Khao Ang Rue Nai Wildlife Sanctuary, Thailand. Canid include larger sample sizes and variation in pack compos- News, http://www.canids.org/canidnews/15/Camera_trap_records_ ition may provide additional insights into reproductive of_dholes_in_Thailand.pdf. mechanisms of this endangered canid. Maia OB, Gouveia AMG (2002) Birth and mortality of maned wolves Chrysocyon brachyurus (Illiger, 1811) in captivity. Braz J Biol 62: Acknowledgements 25–32. The authors thank staff from the Chiang Mai Night Safari Maisch H (2010) The influcence of husbandry and pack management and Chiang Mai Zoo for assisting with sample collection. on dhole, Cuon alpinus reproduction. Int Zoo Yearb 44: 149–164. We are also grateful to Nicole Presley and staff of the endo- Martin P, Bateson P (2007) Measuring Behavior: an Introductory Guide. crinology laboratory of the Faculty of Veterinary Medicine, Cambridge: Cambridge University Press. Chiang Mai University for technical support. Monfort SL, Wasser SK, Mashburn KL, Burke M, Brewer BA, Creel SR (1997) Steroid metabolism and validation of noninvasive endo- Funding crine monitoring in the African wild dog (Lycaon pictus). Zoo Biol This work was supported by the Smithsonian Undersecretary 16: 533–548. for Science’s Endowment Fund [USS-2007]. Munro CJ, Stabenfeldt GH, Cragun JR, Addiego LA, Overstreet JW, Lasley BL (1991) The relationship of serum estradiol and progester- References one concentrations to the excretion profiles of their major urinary metabolites as measured by enzyme-immunoassay and radio- Asa C, Valdespino C (1998) Canid reproductive biology: an integration of immunoassay. Clin Chem 37: 838–844. proximate mechanisms and ultimate causes. Am Zool 38: 251–259. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Paulraj S, Sundararajan N, Manimozhi A, Sally W (1992) Reproduction Songsasen N, Rodden M, Brown JL, Wildt DE (2006) Patterns of fecal of the Indian wild dog (Cuon alpinus) in captivity. Zoo Biol 11: gonadal hormone metabolites in the maned wolf (Chrysocyon bra- 235–241. chyurus). Theriogenology 66: 1743–1750. Rodden MD, Sorenson LG, Sherr A, Kleiman DG (1996) Use of behav- Valdespino C, Asa CS, Bauman JE (2002) Estrous cycle, copulation, and ioral measures to assess reproductive status in maned wolves. Zoo pregnancy in the fennce fox (Vulpes zerda). J Mammal 83: 99–109. Biol 156: 565–585. Velloso A, Wasser SK, Monfort SL, Dietz JM (1998) Longitudinal fecal Rudert S, Brown JL, Ganslober U, Mobius G, Songsasen N (2011) steroid excretion in maned wolves (Chrysocyon brachyurus). Gen Activity pattern, reproductive behaviors and gonadal hormones in Comp Endocrinol 112: 96–107. the raccoon dog (Nyceteutes procyonoides). Zoo Biol 30: 134–148. Walker SL, Waddell WT, Goodrowe KL (2002) Reproductive endocrine Schwarzenberger F, Mostl E, Palme R, Bamberg E (1996) Faecal steroid patterns in captive female and male red wolves (Canis rufus) analysis for non-invasive monitoring of reproductive status in assessed by fecal and serum hormone analysis. Zoo Biol 21: farm, wild and zoo animals. Anim Reprod Sci 42: 515–526. 321–335. Seal US, Plotka ED, Mech D, Packard JM (1987) Seasonal metabolites Wildt DE, Panko WB, Chakraborty P, Seager SWJ (1979) Relationship of and reproductive cycles in wolves. In H Frank, eds, Man and Wolf: serum estrone, estradiol-17β and progesterone to LH, sexual behav- Advances, Issues and Problems in Captive Wolf Research. Dordrecht, ior and time of ovulation in the bitch. Biol Reprod 20: 648–658. The Netherlands: Dr. W. Junk Publisher, pp 109–125. .............................................................................................................................................................. 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Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus)

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Volume 5 � 2017 10.1093/conphys/cox001 Research article Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus) 1 2 3 1 Jaruwan Khonmee , Suvichai Rojanasthien , Chatchote Thitaram , Jureerat Sumretprasong , 4 5 6, Anurut Aunsusin , Chawin Chaisongkram and Nucharin Songsasen Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Department of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Department of Companion Animal and Wildlife Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai 50100, Thailand Chiang Mai Night Safari, Hang Dong, Chiang Mai 50230, Thailand Chiang Mai Zoo, Chiang Mai 50200, Thailand Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA *Corresponding author: Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA 22630, USA. Tel: +1 540 635 0030. Email: songsasenn@si.edu .............................................................................................................................................................. To date, there is no information on reproductive endocrinology of dholes (Cuon alpinus). The objectives of the present study were as follows: (i) to characterize longitudinal profiles of gonadal steroids; and (ii) to examine the relationship between gonadal hormones and sexual behaviours in dholes. Three breeding pairs and two bachelor males were included in the study. Among these, four animals (2 males and 2 females; 4 years old) were imported from The Netherlands to Thailand 3 months before the study onset; the remaining individuals (3 males and 1 female; 5–7 years old) were native born. Faecal samples were collected 3–7 days/week for 12 months, extracted and assessed for gonadal hormone metabolites using a validated enzyme immunoassay. Observations of behaviour were conducted in 30 min sessions, 3–5 days/week. For the three breeding males, testosterone was elevated (P < 0.05) from October to January in the two imported males, whereas the concentration of steroid metabolites was high from April to June and from September to November in the native male. However, there was no clear seasonal pattern of reproductive hormone in the bachelor group. Oestrogen metabolite level of imported females was elevated for 9–12 days in January, followed by a rise in progestagen concentration. For native females, oestrogen metabolites were above the basal values in April and September, each of which was followed by a rise in progestagen concentration that remained elevated for 77 and 112 days, respectively. Sexual behaviours, including solicitation, mount- ing and copulations, were observed during the oestrogen peak in all females. Our findings indicate that reproductive sea- sonality of dholes may depend on the animals’ origin and social group. Key words: Dhole, gonadal steroids, non-invasive hormone monitoring, sexual behaviour Editor: Steven Cooke Received 10 October 2016; Revised 26 December 2016; Editorial Decision 2 January 2017; accepted 9 January 2017 Cite as: Khonmee J, Rojanasthien S, Thitaram C, Sumretprasong J, Aunsusin A, Chaisongkram C, Songsasen N (2017) Non-invasive endocrine monitoring indicates seasonal variations in gonadal hormone metabolites in dholes (Cuon alpinus). Conserv Physiol 5(1): cox001; doi:10.1093/ conphys/cox001. .............................................................................................................................................................. .............................................................................................................................................................. © The Author 2017. Published by Oxford University Press and the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Introduction Materials and methods The dhole or Asiatic wild dog (Cuon alpinus) is a medium- Animals and collection of samples sized, social canid. The species currently inhabits a fragmen- A total of five males and three females maintained in two zoos ted range in southern China, India, Myanmar, Thailand, in Chiang Mai, Thailand (18° 47′ 25″N, 98° 58′ 54″E) were Laos, Vietnam, Malaysia and Indonesia (Durbin et al., included in the study. The three breeding pairs were housed at 2004). Dholes are listed as ‘endangered’ by the International the Chiang Mai Night Safari, two (4 years old) of which were Union for Conservation of Nature (IUCN, 2016), mainly imported from The Netherlands 3 months before the study because of threats associated with habitat fragmentation, onset, and were kept off exhibit in a 3 m × 4 m (width × hunting, prey depletion, competition with other large carni- length) outdoor enclosure. During the study period, the two vores (i.e. tigers and leopards) and diseases transmitted by imported females gave birth; one had one pup that died soon feral/domestic dogs (Durbin et al., 2004). It has been esti- after birth at the end of March and another had a litter of mated that there are <2500 mature individuals living the four pups at the beginning of April, one of which survived wild (IUCN, 2016). The dhole is secretive and lives in highly (hand rearing). The remaining pair (7 years old), born in social, close-knit packs of three to 20 individuals, with a Thailand were maintained on exhibit in a 5 m × 4 m (width × structure of rigid dominance hierarchies (Durbin et al., length) outdoor enclosure. The two bachelor males (5 years 2004). Dholes hunt co-operatively and maintain communica- old) were born in Thailand and kept on exhibit 3 m × 4m tion with pack members by whistling as they move through (width × length) at Chiang Mai Zoo. All animals received dense forests (Fox, 2004). natural light and were fed fresh chicken once daily. Fresh fae- cal samples (~30 g) were collected 3–7 days/week for males To date, there is limited information on dhole reproduc- and 5–7 days/week for females for 12 months. All samples tion. Behavioural data gleaned from a 4 year study in India were stored at −20°C until hormone extraction and analysis. suggested that dholes are seasonal breeders (Paulraj et al., All animal procedures were approved by the National Zoo’s 1992). However, the time of breeding season varies among Institutional Animal Care and Use Committee. regions. Paulraj et al. (1992) reported that breeding occurs between August and December in India. However, a study Faecal extraction conducted in the same country revealed that mating occurs between November and April, with peak activity observed All chemicals were obtained from Sigma Chemical Company during December and January (Durbin et al., 2004). Finally, (St Louis, MO, USA), unless otherwise stated. Frozen faecal dholes are thought to mate mainly during January and May samples were thawed at room temperature, and 0.5 g of each in East Java (Durbin et al., 2004), and those housed in a zoo well-mixed wet sample was placed in a glass tube containing in Germany breed in January (Maisch, 2010). The oestrous 90% ethanol, vigorously shaken in a Multi Pulse vortexer period lasts 14–39 days, and sexual behaviours, including (Glas-Col, Terre Haute, IN, USA) for 30 min, and centri- solicitation, copulatory tie and back-to-back posture have fuged at 1300 g for 20 min. The supernatants were stored at been observed during this period, as in other canids; the −20°C until further analysis. Extraction efficiencies, deter- copulation period ranged from 1 to 9 days (Durbin et al., mined by addition of 2500 d.p.m. H-steroid before extrac- 2004). Based on behavioural observations, dholes exhibited tion were 96.2% [coefficient of variance (CV) <10%] for seasonal polyoestrus with a period of 4–6 weeks between testosterone (male samples) and 88.9% (CV <10%) for pro- cycles (Durbin et al., 2004), which is different from other gesterone (female samples). wild canids, such as the maned wolf (Chrysocyon bra- chyurus; Velloso et al., 1998; Songsasen et al., 2006), Enzyme immunoassay African wild dog (Lycaon pictus; Monfort et al., 1997) and red wolf (Canis rufus; Walker et al., 2002). The gestation Testosterone, oestrogen and progestagen metabolites were period is ~9 weeks (Durbin et al., 2004; Fox, 2004), and lit- quantified by enzyme immunoassay. Antibodies for testoster- ter size ranges from four to 12 pups (Durbin et al., 2004). one (polyclonal testosterone R156/7; 1:8500 dilution), oestro- gen (polyclonal estrone conjugate ECR522; 1:20 000 dilution) To date, there is no information on reproductive endocrin- and progesterone (monoclonal pregnane CL425; 1:10 000 ology of the dhole. Therefore, the main goal of the present dilution) were obtained from the University of California, study was to characterise longitudinal gonadal steroid profiles Davis, CA, USA. Cross-reactivities of R156/7 and ECR522 using a non-invasive endocrine monitoring technique devel- have previously been reported by Munro et al. (1991), oped for domestic and wildlife species (Schwarzenberger et al., whereas that of CL425 was reported by Graham et al. (2001). 1996; Brown, 1995, 2011). The specific objectives were as fol- lows: (i) to determine the influence of seasonality on gonadal Serial dilutions of pooled faecal extracts produced dis- hormone excretion; and (ii) to examine the relationship placement curves parallel to those of the appropriate stan- between reproductive hormones and sexual behaviours in dards. Recovery of added standard to pooled faecal extracts females. demonstrated significant (P < 0.05) recovery (testosterone, .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. y = 0.77x + 39.47, R = 0.997; oestrogen, y = 1.16x + Oestrogen and progestagen metabolites 0.28, R = 0.998; and progestagen, y = 0.79x + 2.30, Longitudinal profiles of oestrogen and progestagen metabo- R = 0.998). Inter-assay CVs were <15% and intra-assay lites were aligned to the day of the oestrogen peak (day 0). CVs <10%. Assay sensitivities were 2.3, 1.95 and 0.78 pg/ Day 0 was identified as the first day that oestrogen rose well for testosterone, oestrogen and progestagen analyses, above baseline concentration by 2 SD (Songsasen et al., respectively. 2006). Baseline values for each individual were calculated by an iterative process, whereby high values (exceeding the High-performance liquid chromatography mean plus 1.5 SD) were excluded. Each time the average was recalculated, and the elimination process repeated until no The quantities and relative proportions of immunoactive values exceeded the mean plus 1.5 SD. Data were reported as testosterone, estrogen and progestagen metabolites in dhole means ± SEM. Longitudinal steroid metabolite profiles were faecal extracts were determined using reverse-phase high-per- divided into three reproductive stages using modified criteria formance liquid chromatography (HPLC; Microsorb C-18 of those described by Velloso et al. (1998) and Walker et al. Column, Rainen Inc., Woburn, MA, USA). For each sex, five (2002), as follows: (i) anoestrus (days −30 to −11 before the faecal extracts were combined, air dried, resuspended in 1 ml oestrogen peak); (ii) pro-oestrus (days −10 to −1); (iii) oestrus methanol, dried under air and stored at −20°C until analysis. (days 0–6); and (iv) dioestrus (days 7–93 or until the end of Extract pools were reconstituted with 0.5 ml phosphate- faecal collection in the two imported females). Differences in buffered saline and filtered through a C-18 spice cartridge faecal steroid concentration among different stages of repro- (VWR, West Chester, PA, USA), eluted with 5 ml methanol, ductive cycles (females) within the same individuals were air dried and spiked with known radio-labelled steroids ( H- 3 3 3 determined by ANOVA followed by the Holm–Sidak meth- testosterone, H-estradiol-17β, H-estrone, H-estrone-sulfate od for multiple comparison. Differences were considered sig- and H-progesterone, ~2500 d.p.m. each). Subsequently, the nificant when the P-value was <0.05. samples were dried down and resuspended in 300 µl metha- nol. Testosterone metabolites were separated using a gradient of 45% methanol over 90 min (1 ml/fraction; 1 ml/min flow Results rate). Oestrogen metabolites were separated using a 20–80% methanol gradient over 80 min, and progestagen metabolites High-performance liquid chromatography were separated using 20–30–50–100% acetonitrile gradients over 15, 45 and 60 min, respectively. Co-elution profiles of the Evaluation of faecal extracts by HPLC revealed the presence respective radio-labelled steroids in each HPLC run were deter- of several testosterone metabolites, one (10%) of which mined by adding 100 µlofeachHPLC fractionto 3ml of exhibited similar retention time to testosterone (fractions scintillation fluid (Ultima Gold; Packard, Meriden, CT, USA) 30–35; Fig. 1). The HPLC analysis of oestrogen metabolites and counted in a dual-label channel β scintillation counter demonstrated three immunoreactive peaks (fractions 15–20, (Beckman, Fullerton, CA, USA). Each of the remaining HPLC 57–61 and 60–63), which co-eluted with estrone-3-sulfate, fractions was then air dried, reconstituted in 0.2 ml assay buffer estrone and estradiol (Fig. 2). Progestagen immunoreactivity and quantified for immunoactivity by enzyme immunoassay. was associated with a single peak (fractions 63–68), which corresponded to the radio-labelled progesterone (Fig. 3). Behavioural observations Steroid metabolite concentrations of male Quantitative behavioural data were collected 3–5 days/week in all breeding females during 30 min observation sessions con- dholes ducted in the morning with instantaneous scan sampling Mean ± SEM testosterone metabolites of imported (breed- (Martin and Bateson, 2007). During the observation period, all ing), native (breeding) and native (bachelor) males are shown sexual behaviours, including solicitation, mounting and copu- in Fig. 4A–C. For breeding males, testosterone concentra- lation, were recorded using an ethogram adapted from that tions varied among months. However, there were individual developed for the maned wolf (Rodden et al., 1996). The differences in the time during which high testosterone con- behavioural data were then superimposed on endocrine profiles. centrations were observed. Specifically, testosterone metabol- ite concentrations of the two imported dholes (males A and Hormonal profiles and data analysis B) were at low levels during February–September. Steroid concentrations significantly increased in October and remained Testosterone elevated (P < 0.05) until January (Fig. 4A). Mean testosterone metabolites were calculated on a monthly basis and compared across months within each male to For the native male that was paired with a female (male C), determine the influence of seasonality on hormone produc- testosterone metabolite concentrations were low in February, tion using Kruskal–Wallis one-way analysis of variance slightly elevated in April–June, significantly declined in July (ANOVA) followed by Dunn’s method (SigmaStat 3.0; SPSS and August and then substantially increased in September and Inc., Chicago, IL, USA). Significance was set at 95%. remained at high concentrations until November (P < 0.05; .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Immunoactivity (ng/ml) Immunoactivity (ng/ml) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Testosterone Radioactivity 16000 3500 Immunoactivity 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 HPLC fractions Figure 1: Co-chromatographic high-performance liquid chromatography separation of faecal testosterone metabolites of male dholes. Radioactivity Estrone Estradiol 9000 40 Immunoactivity Estrone-3- sulfate 0 0 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 HPLC fractions Figure 2: Co-chromatographic high-performance liquid chromatography separation of faecal oestrogen metabolites of female dholes. Fig. 4B). Seasonal variations in testosterone concentrations during oestrus than in pro-oestrus, dioestrus and anoestrus. were not apparent in the two bachelor males (Fig. 4C). Progestagen metabolite concentrations were at the baseline level during pro-oestrus, slightly increased during oestrus, sig- nificantly elevated during dioestrus (P < 0.05) and returned to Steroid metabolite concentrations and the baseline levels during the anoestrous period. reproductive behaviours of female dholes In all females, the increase in oestrogen coincided with vul- Faecal oestrogen and progestagen concentrations during val swelling and sexual behaviours, including solicitation, various stages of the reproductive cycle are shown in Fig. 5. mounting and copulations (Fig. 6A–C). However, there were Regardless of the pregnancy outcome, all females exhibited a marked differences in reproductive season between imported similar endocrine pattern. Specifically, mean oestrogen metab- and native females. The oestrogen peak of the imported females olite concentrations in all females were higher (P < 0.05) was observed in January, followed by a rise in progestagen .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Radioactivity (cpm/ml) Radioactivity (cpm/ml) Immunoactivity (ng/ml) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. Progesterone Radioactivity Immunoactivity 7000 4000 0 0 1 112131415161718191 101 111 HPLC fractions Figure 3: Co-chromatographic high-performance liquid chromatography separation of faecal progestagen metabolites of female dholes. concentrations, which remained elevated until the beginning of that reported in the raccoon dog (Nyctereutes procyonoides; April (Fig. 6A and B). In the native pair, copulations were Rudert et al., 2011). Given that the majority of metabolites observed in April and September, but pups were not produced. were not associated with testosterone, this assay could not Two oestrogen peaks, 149 days apart, were observed in the be used as an exclusive index for this steroid hormone. native female (in April and September); each was followed by Nevertheless, variations in testosterone metabolite concentra- increased progestagen metabolite concentrations, which tions among seasons with high hormone concentrations remained elevated for 77 and 114 days, respectively (Fig. 6C). observed during breeding periods in paired individuals indi- cated that this assay can be used to assess gonadal functions in male dholes. Unlike the African wild dog (Monfort et al., 1997), red wolf (Walker et al., 2002) and maned wolf Discussion (Velloso et al., 1998), which excrete mostly estradiol and Non-invasive faecal hormone metabolite monitoring has estrone, dhole samples contained a significant amount of proved to be a valuable tool in advancing the understanding estrone-3-sulfate (20%) in addition to the two metabolites of reproductive functions and enhancing ex situ management found in other canids. The immunoreactive progestagen of various wildlife species, including carnivores (Brown et al., peak of dhole samples was co-eluted with progesterone, 1995). The present study characterized gonadal hormone pro- which also differed from the findings in the African wild dog files in male and female dholes and investigated the influence (Monfort et al., 1997), red wolf (Walker et al., 2002) and of seasonality on the steroid excretion patterns in this species. maned wolf (Velloso et al., 1998). In these three canids, pro- Our data demonstrated that dholes are seasonal breeders, with gestagen metabolites excreted in faecal samples were less gonadal hormone concentrations that varied among months, polar than progesterone. Given that increases in both oestro- especially in breeding pairs. Specifically, in imported males, tes- gen and progestagen concentrations coincided with reproduct- tosterone metabolites were elevated from October to January ive behaviours observed in the three pairs, these assays can be for the males, whereas steroid metabolites in the breeding used to evaluate reproductive functions in female dholes. native male were at a high level during April–June and To date, information regarding dhole reproductive biology September–November. The seasonal pattern of testosterone has been generated exclusively from behavioural observation excretion corresponded to sexual behaviours and increased of captive and wild individuals (Durbin et al., 2004; Maisch, gonadal function in the females. Our findings also suggested 2010). It has been suggested that female dholes exhibit sea- that intrinsic (e.g. genetic) factors, rather than environmental sonal polyoestrus with 4–6 weeks between the end of the first conditions, may influence the time during which reproduction and beginning of the next cycle (Durbin et al., 2004). In the occurs. Finally, our data suggested that the dhole is a polyoes- present study, we observed that the non-pregnant female trous canid, a characteristic that is unique among species (female C) exhibited two estrous cycles during the study peri- within the Canidae family. od, one from April to June and one from September to In the present study, it was found that only a small pro- December, whereas the two pregnant females cycled only once portion (10%) of androgen metabolites were excreted as tes- a year. Although our observation was based on one female, it tosterone in dhole faecal samples. This finding is similar to appeared that our endocrine data confirmed behavioural .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Radioactivity (cpm/ml) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Imported (breeding) male A Imported (breeding) male B ** ## ** ## ** ** ## 16000 b ab ab ab b ab ab a a Feb Mar April May June July Aug Sep Oct Nov Dec Jan Feb Native (bachelor) male D Native (bachelor) male E ** ** Figure 4: Mean ± SEM testosterone metabolite concentrations of two imported males housed in breeding pairs (A), a native male housed in a **, ##, ++ a,b breeding pair (B) and two native males housed as a bachelor group (C). Symbols or superscript letters indicate significant differences among months within the same male. P < 0.05. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Faecal testosterone metabolites Faecal testosterone metabolites Faecal testosterone metabolites (ng/g wet faeces) (ng/g wet faeces) (ng/g wet faeces) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. which was consistent with the oestrous period observed in Imported (breeding) female A Oestrogen the females. Given that the two imported pairs gave birth 10000 Progestagen in March and April, we estimated that breeding occurred in January, during the first reproductive season after they were transported to Thailand, and this pattern remained the same in the following year. This breeding period (January) differed from that observed in the native pair and wild counterparts living in Thailand (April and September). Specifically, the native pair exhibited breeding behaviours in April and ab ab a bc September, which coincided with increased gonadal metabol- b c b 0 ite concentrations in both male and female. A camera-trap Pro-oestrus Oestrus Dioestrus Anoestrus study conducted in eastern Thailand documented a photo- graph of two juveniles (~6 months old) in May, indicating Imported (breeding) female B that breeding probably occurred in September or October in Oestrogen wild individuals (Jenks et al.,2012). Furthermore, a lactating Progestagen female was captured in a Thai protected area in mid- 8000 February, suggesting that this individual probably bred in late October to early November (N.S., personal observation). Thus, it appears that the reproductive season of imported dholes in the present study did not shift to the same period as that of native animals, even after being in Thailand for 1 year. ab This is in contrast to observations in the maned wolf. The ab b b b maned wolf breeding season differs by 6 months between the Northern and Southern hemispheres, with the peak breeding Pro-oestrus Oestrus Dioestrus Anoestrus occurring in October (autumn) and April (autumn) in North 12000 and South America, respectively (Maia and Gouveia, 2002). Oestrogen Native (breeding) female C Analysis of faecal testosterone concentrations of a male maned a Progestagen wolf transported from Brazil to the USA in June (i.e. the end of breeding season in Brazil) showed that this individual adapted 8000 ab to the reproductive season of North America shortly after being placed in the new environment, as steroid concentrations remained elevated until the following January (i.e. the end of the breeding season in the USA; N.S., unpublished data). Thus, bc 2000 it appears that reproductive seasonality in the maned wolf is ab b ab regulated by environmental factors (photoperiod). The influence of photoperiod on reproduction has also been observed in the Pro-oestrus Oestrus Dioestrus Anoestrus red wolf (Canis rufus; Walker et al., 2002), in which variation in faecal androgen metabolite concentrations is linked to chan- Figure 5: Mean ± SEM concentrations of ovarian steroid metabolites during pro-oestrus (days −10 to −1 of the oestrogen peak), oestrus ging day length; steroid concentration increases in late autumn (days 0–6), dioestrus (days 7–93) and anoestrus (days −30 to −11) of (October) and peaks in winter (February). Given that imported the two imported females (A and B) and the native female (C). dholes maintained the same reproductive seasonal pattern abc Different superscript letters indicate significant differences among they had exhibited in Europe even after living in Thailand for reproductive cycles within the same individual for each steroid 1 year, we suspect that the reproductive season of this species hormone (P < 0.05). may be regulated by other factors, rather than the environment. Based on morphology, geographical locations and genetic observations of others (Durbin et al.,2004), suggesting that information, dholes are classified into 11 subspecies (Durbin dholes are unique amongst wild canids, which are seasonally et al., 2004; Iyengar et al., 2005). Recently, analysis of mito- monoestrous (Asa and Valdespino, 1998). Future studies that chondrial DNA of imported and native individuals included include multiple females are required to confirm this unique in the present study revealed that these two animal groups reproductive characteristic of the dhole. differed in the haplotype of mitochondrial control region Another interesting aspect is that there appeared to be dif- (J. Kayman, personal communication). Specifically, the for- ferences in reproductive season between imported and native mer was classified as having haplotype R, the type found individuals, at least during the study period (1 year). In the mostly in captive dholes housed in European zoos. However, imported males (males A and B), testosterone metabolites all native individuals were of haplotype U, a novel haplotype reached peak concentrations between October and January, that has not been previously described (Iyengar et al., 2005). .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Gonadal hormone metabolites Gonadal hormone metabolites Gonadal hormone metabolities (ng/g wet faeces) (ng/g wet faeces) (ng/g wet faeces) Faecal Progestagen (ng/g wet faeces) Faecal progestagen (ng/g wet faeces) Faecal Progestagen (ng/g wet faeces) Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. 3500 39000 Oestrogen Progestagen Swollen vulva 2500 Elevated Testosterone Birth 0 0 Date Oestrogen 3500 39000 Progestagen Swollen vulva Elevated Testosterone Birth 0 0 Date Oestrogen 3500 Progestagen 39000 Mounting Copulations Elevated Testosterone 0 0 Date Figure 6: Longitudinal profile of gonadal steroids of the two imported females (A and B) and the native female (C) overlaid with reproductive events/behaviours and the interval during which elevated testosterone metabolites were observed in the respective male. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Faecal Oestrogen (ng/g wet faeces) Faecal Oestrogen (ng/g wet faeces) Faecal Oestrogen metabolites (ng/g wet faeces) Conservation Physiology � Volume 5 2017 Research article .............................................................................................................................................................. Asher GW, O’Neill KT, Scott LC, Mockett BG, Fisher MW (2000) Genetic Thus, it is possible that the difference in seasonal cyclicity influence on reproduction in female red deer (Cervus elaphus): sea- between imported and native dholes may be, in part, asso- sonal luteal cyclicity. Anim Reprod Sci 28: 43–59. ciated with genetic divergence. The influence of genetic fac- tors on reproductive cyclicity has been previously shown in Brown JL (2011) Female reproductive cycle of wild female felids. Anim the red deer (Cervus elaphus; Asher et al., 2000). Reprod Sci 124: 155–162. By combining behavioural observation and endocrine moni- Brown JL, Wemmer CM, Lehnhardt J (1995) Urinary cortisol analysis toring, we have demonstrated that the dhole oestrous period for monitoring adrenal activity in elephants. Zoo Biol 14: 533–542. ranges from 9 to 13 days, which is similar to that reported in Concannon PW, Hansel W, McEntree K (1977) Changes in LH, proges- other canids, including the grey wolf (Canis lupus; Seal et al., terone and sexual behaviour associated with preovulatory luteini- 1987), fennec fox (Vulpes zerda; Valdespino et al.,2002), red zation in the bitch. Biol Reprod 17: 604–613. wolf (Walker et al.,2002), maned wolf (Songsasen et al.,2006) and domestic dog (Edqvist et al.,1975; Concannon et al., Durbin LS, Venkataraman A, Hedges S, Duckworth W (2004) Dhole 1977; Wildt et al.,1979). Behavioural studies conducted on (Cuon alpinus). In C Sillero-Zubiri, M Hoffman, DW Macdonald, eds, captive dholes reported that the oestrous period is 3–4 weeks, Canids: Foxes, Wolves, Jackals and Dogs. Gland, Switzerland: IUCN, and the copulation period ranges from 1 to 9 days (Paulraj pp 201–218. et al.,1992; Maisch, 2010). Courtship behaviours of dhole observed in the present study were similar to those reported in Edqvist LE, Jaohansson EDB, Kasström H, Olsson SE, Richkind M (1975) the previous study (Paulraj et al.,1992); these include mount- Blood plasma levels of progesterone and oestradiol in the dog ing, copulatory tie and back-to-back copulatory posture. In the during the oestrous cycle and pregnancy. Acta Endocrinol 78: present study, two oestrogen peaks 149 days apart were 554–564. observed in the native female; each was followed by increased Fox MW (2004) The Whistling Hunters: Field Studies of the Asiatic Wild progestagen metabolite concentrations that remained elevated Dog (Cuon alpinus). Albany: State University of New York Press. for 77 and 114 days, respectively. Given that neither reproduct- ive cycle resulted in pregnancy, the discrepancy in the length of Graham LH, Schwarzenberger F, Mostl E, Galama W, Savage A (2001) the dioestrous period remains to be elucidated. A versatile enzyme immunoassay for the determination of proges- tagens in feces and serum. Zoo Biol 20: 227–236. In summary, by using non-invasive hormone monitoring, IUCN (2016). The IUCN Red List of Threatened Species. Version 2016-3. we have established the first endocrine database characteris- www.iucn4redlist.org. ing the reproductive cycle of the endangered dhole. Despite the small sample size, longitudinal faecal steroid monitoring Iyengar A, Babu VN, Hedges SA, Venkataraman B, Maclean N, Morin through enzyme immunoassay assessments has revealed sev- PA (2005) Phylogeography, genetic structure, and diversity in the eral reproductive characteristics of this understudied canid, dhole (Cuon alpinus). Mol Ecol 14: 2281–2297. including the evidence of the following: (i) seasonal polyoes- trus in the females; and (ii) the potential of genetic impact on Jenks KE, Songsasen N, Leimgruber P (2012) Camera trap records of timing of reproductive seasonality. Future studies that dholes in Khao Ang Rue Nai Wildlife Sanctuary, Thailand. Canid include larger sample sizes and variation in pack compos- News, http://www.canids.org/canidnews/15/Camera_trap_records_ ition may provide additional insights into reproductive of_dholes_in_Thailand.pdf. mechanisms of this endangered canid. Maia OB, Gouveia AMG (2002) Birth and mortality of maned wolves Chrysocyon brachyurus (Illiger, 1811) in captivity. Braz J Biol 62: Acknowledgements 25–32. The authors thank staff from the Chiang Mai Night Safari Maisch H (2010) The influcence of husbandry and pack management and Chiang Mai Zoo for assisting with sample collection. on dhole, Cuon alpinus reproduction. Int Zoo Yearb 44: 149–164. We are also grateful to Nicole Presley and staff of the endo- Martin P, Bateson P (2007) Measuring Behavior: an Introductory Guide. crinology laboratory of the Faculty of Veterinary Medicine, Cambridge: Cambridge University Press. Chiang Mai University for technical support. Monfort SL, Wasser SK, Mashburn KL, Burke M, Brewer BA, Creel SR (1997) Steroid metabolism and validation of noninvasive endo- Funding crine monitoring in the African wild dog (Lycaon pictus). Zoo Biol This work was supported by the Smithsonian Undersecretary 16: 533–548. for Science’s Endowment Fund [USS-2007]. Munro CJ, Stabenfeldt GH, Cragun JR, Addiego LA, Overstreet JW, Lasley BL (1991) The relationship of serum estradiol and progester- References one concentrations to the excretion profiles of their major urinary metabolites as measured by enzyme-immunoassay and radio- Asa C, Valdespino C (1998) Canid reproductive biology: an integration of immunoassay. Clin Chem 37: 838–844. proximate mechanisms and ultimate causes. Am Zool 38: 251–259. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018 Research article Conservation Physiology � Volume 5 2017 .............................................................................................................................................................. Paulraj S, Sundararajan N, Manimozhi A, Sally W (1992) Reproduction Songsasen N, Rodden M, Brown JL, Wildt DE (2006) Patterns of fecal of the Indian wild dog (Cuon alpinus) in captivity. Zoo Biol 11: gonadal hormone metabolites in the maned wolf (Chrysocyon bra- 235–241. chyurus). Theriogenology 66: 1743–1750. Rodden MD, Sorenson LG, Sherr A, Kleiman DG (1996) Use of behav- Valdespino C, Asa CS, Bauman JE (2002) Estrous cycle, copulation, and ioral measures to assess reproductive status in maned wolves. Zoo pregnancy in the fennce fox (Vulpes zerda). J Mammal 83: 99–109. Biol 156: 565–585. Velloso A, Wasser SK, Monfort SL, Dietz JM (1998) Longitudinal fecal Rudert S, Brown JL, Ganslober U, Mobius G, Songsasen N (2011) steroid excretion in maned wolves (Chrysocyon brachyurus). Gen Activity pattern, reproductive behaviors and gonadal hormones in Comp Endocrinol 112: 96–107. the raccoon dog (Nyceteutes procyonoides). Zoo Biol 30: 134–148. Walker SL, Waddell WT, Goodrowe KL (2002) Reproductive endocrine Schwarzenberger F, Mostl E, Palme R, Bamberg E (1996) Faecal steroid patterns in captive female and male red wolves (Canis rufus) analysis for non-invasive monitoring of reproductive status in assessed by fecal and serum hormone analysis. Zoo Biol 21: farm, wild and zoo animals. Anim Reprod Sci 42: 515–526. 321–335. Seal US, Plotka ED, Mech D, Packard JM (1987) Seasonal metabolites Wildt DE, Panko WB, Chakraborty P, Seager SWJ (1979) Relationship of and reproductive cycles in wolves. In H Frank, eds, Man and Wolf: serum estrone, estradiol-17β and progesterone to LH, sexual behav- Advances, Issues and Problems in Captive Wolf Research. Dordrecht, ior and time of ovulation in the bitch. Biol Reprod 20: 648–658. The Netherlands: Dr. W. Junk Publisher, pp 109–125. .............................................................................................................................................................. Downloaded from https://academic.oup.com/conphys/article-abstract/5/1/cox001/2996800 by Ed 'DeepDyve' Gillespie user on 07 June 2018

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Conservation PhysiologyOxford University Press

Published: Feb 15, 2017

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