Faecal glucocorticoid analysis as indicator of stress and its effect on the reproduction hormones of the female Malayan sun bear (Helarctus malayanus) in captivity.

Faecal glucocorticoid metabolite (FGM) is frequently used as a non-invasive tool to assess adrenocortical activity in animal conservation and welfare studies. However, studies assessing the stress level and its effect on the reproductive hormones of female Malayan sun bear is lacking. This study aims to assess the level of stress of the female Malayan sun bear in captivity and its effect on the female reproductive hormones of the female Malayan sun bears in captivity in Malaysian Borneo. Through FGM analysis via the enzyme-linked immunoassay (ELISA) method, it was found that the bear living in BSBCC has the lowest level of glucocorticoid. as for the relationship between the level of stress and the female reproductive hormones, the level of estrogen which was indicated by estradiol, decreases as the level of FGM increases. In contrast, the level of progesterone increase as the level of FGM increases. It can be concluded that the level of stress in captivity could affect the level of estrogen and progesterone and a high level of progesterone does not always indicate a good reproductive function in female mammals.


Introduction
In conservation management of endangered creature such as the Malayan sun bear, a widely adopted strategy involves the practice of keeping them in captive institution, such as zoos, as part of a breeding initiative.This approach serves as a preventive measure against the potential decline of the wild population.Through implementation of the breeding programs, we can effectively preserve genetic diversity and facilitate the reintroduction of this endangered species back into its natural habitat.
In captive institution, the bear receives veterinary attention and is provided with sufficient nourishment [1].Numerous studies have indicated that captive populations generally exhibit higher rates of survival and reproduction compared to their wild counterparts [2].However, it is crucial to acknowledge that captivity differs significantly from the natural habitat of these animals [3] giving rise to various challenges, including the manifestation of anomalous behaviours, such as stereotypic behaviours.These behaviours may be used as indicators of compromised animal welfare [4] since the ability to express innate behaviours is limited [1].The frustration and stress stemming from their inability to engage in species-specific behaviours disrupts their well-being and alters their behavioural 1316 (2024) 012001 IOP Publishing doi:10.1088/1755-1315/1316/1/012001 2 patterns, potentially leading to the emergence of stereotyped behaviours [5].This issue has drawn considerable attention in recent decades [6,7,8].
The presence of stress in captive environments, as indicated by glucocorticoid levels, may impede conservation efforts, including captive breeding [9].The levels of glucocorticoids may have repercussion against the female reproductive hormones specifically, progesterone and oestrogen.The influence of glucocorticoid levels on progesterone hormones is evident in studies conducted by [10] and [11] on small mammals.Their research underlines the link between irregular oestrous cycles and low progesterone levels and high glucocorticoid levels in the blood of the studied species.
Glucocorticoid hormones do not solely have negative effects on mammalian reproduction.In fact, the absence of glucocorticoids may lead to reduced fertility and disrupt foetal development.In human medicine, glucocorticoids may be administered to pregnant women as a form of treatment for preterm labour, autoimmune disorders such as asthma, or adrenal insufficiency.However, exposure to elevated levels of glucocorticoids induced by stress at the onset of receptivity or during foetal development may potentially weaken the female's immune system, which is necessary for establishing pregnancy and regulating foetal development [9].
The correlation between stress and reproduction in Malayan sun bears has not been fully elucidated [11,12].Hence, the objective of this study is to examine the welfare of sun bears residing in captivity by evaluating their stress levels and assessing the influence of stress on the reproductive hormone levels of female sun bears.

Study site and sample collection
This study was conducted in the Malaysian region of Borneo, encompassing the states of Sabah and Sarawak.In Sabah, faecal samples of Malayan Sun Bears were collected from the Lok Kawi Wildlife Park (LKW) and Borneo Sun Bear Conservation Centre (BSBCC).In Sarawak, samples were obtained from the Matang Wildlife Centre (MWC).Access licences for study and sample collection were provided by Sabah Biodiversity Centre (SaBC) (Reference number: JKM/MBS.1000-2/2JLD.12(66)) and Sarawak Forestry Corporation (SFC) (Reference number: SFC.810-4/6/1 (2022)).Export licence for transporting the samples from Sarawak to Sabah was provided by SFC (Permit number: WL 03/2022) and this study was ethically approved by the Committee of Animal Research Ethics of Universiti Teknologi MARA (UiTM) with the ethics approval number: UiTM CARE: 379/2022.Faecal samples were collected immediately after defecation.In order to preserve the integrity of the samples and halt microbial deterioration that could alter cortisol levels, the samples were stored in 50mL sample bottles and placed in a cooler box equipped with reusable icepacks.These collected samples were obtained for the analysis of faecal glucocorticoids, oestradiol, and progesterone hormones .
The bear keeper at each study site assisted in collecting the faecal sample from the female Malayan sun bear at each location.The bear keeper separated the subject animals into individual cages.The faecal sample collection was done in the morning, before the bear keeper cleaned the cages.The collected sample was then placed in a 50 mL sample bottle, which was stored in a cool box containing a reusable ice pack to store and preserve the samples until reaching laboratory at the UiTM Sabah branch.The samples were then kept in a refrigerator maintained at a temperature of 1℃ until further analysis.

Hormones extractions
The method follows [13].Initially, 0.6 g of faecal sample was placed in a centrifuge tube filled with 2 ml of 90% methanol.The centrifuge tubes were then vortexed for 30 minutes and then centrifuged again for 20 minutes at a speed of 1500 rpm.
Subsequently, the samples were diluted in the ratio of 1:2 serial dilution to extract the hormones cortisol and progesterone from the faecal samples.However, no dilution was carried out for the extraction of the hormone oestradiol, as diluting the sample would result in a lower yield of oestradiol.
The serial dilution was started by extracting 500µl of the supernatant obtained from the initial centrifuged sample.Meanwhile, the second tube was filled with 900µl of Phosphate buffer solution (PBS).The dilution was performed by transferring 100µl of the supernatant from the first diluting tube to the second diluting tube.The diluted supernatants were then stored in a refrigerator at a temperature of -20℃ until further analysis using ELISA kits.

Faecal cortisol, oestrogen and progesterone hormone analysis.
Faecal steroid analysis involved the utilization of specific ELISA kits for the detection individual targeted hormones.The detection of cortisol hormone utilized the COR ELISA Kit (catalog no.EU0391, Finetest, Wuhan, China), the detection of oestradiol hormone used E2 ELISA Kit (catalog no.EU0390, Finetest, Wuhan, China), and detection of progesterone hormone used PG ELISA Kit (catalog no.EU0398, Finetest, Wuhan, China).Each ELISA kit was employed according to the manufacturer's instructions, with specific procedures outlined for each kit.Although the procedures were generally similar for all three ELISA kits, each kit utilized a different type of detection antibody.

Procedure for the Enzyme-Linked Immunoassay (ELISA).
ELISA was done according to the manufacturer's instruction.Briefly, 50µl of the samples placed into microplate wells of each ELISA kit.Subsequently, 50µl of biotinylated detection antibody (Ab) was added to each well.The samples were incubated at 37℃ for a duration of 45 minutes.Following the incubation period, the wells were aspirated, and the washing process done thrice using 350µl of washing buffer.During the final wash, any remaining washing buffer was aspirated, and the microplate wells were inverted and gently patted against a clean absorbent paper.This step ensured complete removal of any liquid from the wells, ensuring optimal performance for the subsequent reading of optical densities.Next, 100µl of HRP-Conjugate was added to each well and incubated at 37℃ for 30 minutes.Once again, the content of the wells was aspirated, and the wells were washed 5 times using the same washing technique.Subsequently, 90µl of Substrate Reagent was added to each well, and the wells were incubated at 37℃ for 15 minutes.Following the incubation with the substrate, 50µl of Stop Solution was added to each well.Finally, the microplate wells were placed in a microplate reader set to a wavelength of 450nm, and the optical density (OD) value of each well was recorded.

Stress level of the female Malayan sun bears in captivity.
The female Malayan sun bears housed in BSBCC exhibited the lowest average cortisol concentration compared to those at the LKW and MWC.The average cortisol concentrations recorded at each site were 33.45 ng/mL, 55.34 ng/mL, and 67.58 ng/mL, respectively (Figure 3.1).As indicated by the oneway ANOVA test, the difference in average cortisol concentration among the three sites are statistically significant (F = 13.08,p < 0.001).Female Malayan sun bears housed at the MWC exhibited the highest average cortisol concentration, suggesting elevated levels of stress.In contrast, the bears at the BSBCC displayed the lowest average cortisol concentration.The size of the bear cage is a potential factor contributing to the higher cortisol levels observed in female Malayan sun bears [14,7].At MWC, the sun bears were housed in cages accommodating either 3 or 5 individuals.LKW maintained 2 to 3 bears per cage, while the BSBCC housed 1 to 3 individuals per cage.The population density of bear cages at BSBCC ranged from 0.17 individuals/m 2 to 0.50 individuals/m 2 .Lok Kawi Wildlife Park had two types of cages, with the first type having a population density of 0.46 individuals/m 2 and the second type having a population density of 0.52 individuals/m 2 .The first type of cage housed 2 bears, while the second type accommodated 3 bears.At the Matang Wildlife Centre, there were three types of cages.The first cage had a population density of 0.67 individuals/m 2 , the second cage had a population density of 0.54 individuals/m 2 , and both cages housed sun bears for open exhibition.The third cage, known as the Night Den, had a population density of 1.02 individuals/m 2 .A higher population density in the bear cages indicates a larger number of individuals living within a single cage.
The stress levels of the sun bears at each study site can be influenced by the population density of the bear cages and the presence of enrichment activities.Bears residing in cages with higher population densities, such as those at the MWC, exhibit the highest average cortisol levels compared to sun bears at LKW and the BSBCC.These findings are aligned with previous research by [13] who observed that enclosure size can impact pacing behaviour and stress levels in captive tigers.Additionally, [7] noted an increase in faecal glucocorticoid levels among animals previously accustomed to living in open spaces when they were confined in captivity.Furthermore, reduced space between individual bears within higher population density cages contributes to elevated faecal glucocorticoid levels.This is particularly significant considering that sun bears in the wild are solitary animals with an average home range ranging from 6.2 km 2 to 20.6 km 2 per individual, and in some cases, reaching up to 130 km 2 [15,16].statistically significant moderate [17] negative correlation between stress levels and oestradiol levels (r = -0.425,n = 22, p = 0.049).Figure 3.2 The relationship between stress level and level of estradiol hormone in female Malayan sun bears in captivity.

The correlation between the level of stress with the level of reproductive hormone in female
Figure 3.3 illustrates the correlation between stress levels (cortisol concentration) and progesterone levels in captive female Malayan sun bears.A Pearson correlation test was performed to examine the relationship between stress levels and progesterone hormone levels.The results revealed a statistically significant strong, positive correlation [17] between stress levels and progesterone levels (r = 0.708, n = 22, p = 0.000).

Figure 3.3 The relationship between stress level and level of progesterone hormone in female Malayan
sun bears in captivity.
The findings from Figure 1.3 and Figure 1.4 demonstrate a negative correlation between oestradiol hormone levels and cortisol concentration, and a positive correlation between progesterone levels and cortisol concentration.These results aligned with a previous study conducted by [18], which observed that Malayan sun bears with high oestrogen levels exhibited lower cortisol levels.Similarly, in our study, we observed a decrease in oestradiol levels as cortisol levels increased.This suggests that the Relationship between stress level (cortisol concentration) and level of progesterone hormone in female Malayan sun bear in captivity stress experienced by female Malayan sun bears in our study may be attributed to agonistic behaviours arising from heightened social influences, as indicated by [18].During the oestrous cycle, oestradiol, the primary type of oestrogen in adult breeding females, is essential for the thickening the uterine lining and prepare for pregnancy.Additionally, during ovulation, there is a peak in oestrogen levels, which triggers the release of the ovum from the ovary.On the contrary, as stress levels increase, the concentration of progesterone do as well.This relationship between stress and progesterone levels aligns with the findings of [18], who reported that high levels of cortisol and progesterone were associated with increased agonistic behaviour.In our study, we identified several factors that contributed to elevated levels of faecal glucocorticoid metabolites (FGMs), including parasite load, reduced home range, and the absence of enrichments in captivity, as supported by [19] and [14].Consequently, heightened stress levels may suppress the production of oestradiol, potentially inhibiting ovulation and hindering efforts for captive breeding [20].
The findings of this study are further supported by [21], who highlighted that elevated levels of progesterone do not always indicate optimal reproductive function in female mammals.In fact, stressrelated high progesterone levels reduce the potential of a successful conception.The host organism's body react by rising its level of the stress hormone glucocorticoid (GC).This response redistributes resources within the body, converting stored carbohydrates into energy for survival and immunological responses, rather than non-essential processes like reproduction.In essence, when confronted with stressful situations or conditions, the organism's physiology prioritizes survival over reproductive capability [22,20,23,24].
Furthermore, it has been observed that stress can impact the hypothalamus, leading to a decrease in the synthesis and secretion of gonadotropin-releasing hormone (GnRH), as well as affecting the anterior pituitary gland, resulting in reduced pituitary responsiveness to GnRH and the modulation of gonadotropin function.This modulation includes an increase in the activity of gonadotropin-inhibitory hormone (GnIH) cells.The suppression of GnRH production ultimately affects the synthesis and release of luteinizing hormone (LH).Therefore, this reduction in LH hormone production results in decreased oestrogen production, providing additional support for the findings illustrated in Figure 4.4 [25,20].
Instead of facilitating the release of gonadotropin-releasing hormone (GnRH), stress in animals triggers the hypothalamus to release adrenocorticotropic hormone (ACTH), which in turn stimulates the adrenal cortex to secrete glucocorticoids.Animals with elevated levels of glucocorticoids experience increased plasma glucose levels due to gluconeogenesis, as well as heightened rates of fat, protein, and carbohydrate metabolism that support the stress response [7,26,27].Consequently, the stress levels in female Malayan sun bears can impact the levels of oestradiol and progesterone hormones.

Conclusion
In conclusion, the average concentration of feacal glucocorticoid metabolites (FGMs), which a measure of stress in captive female Malayan sun bears, was highest in MWC, followed by LKW, and lowest in BSBCC.The reproductive hormones in female Malayan sun bears, notably oestrogen and progesterone, are affected by these stress levels.The relationship between stress level and oestrogen/progesterone levels is statistically significant.The oestrogen level falls when cortisol level rises, but progesterone level rise.Consequently, high levels of stress can influence the reproductive hormone balance in female Malayan sun bears, potentially affecting conservation efforts for this endangered species.Therefore, it is recommended to improve the bear enclosures by providing larger spaces and enrichments to mitigate their stress levels in captivity.Further study is also eminent to perceive a more established correlation between stress and fertility of the female Malayan sun bear.

Figure 3 . 1
Figure 3.1 The average cortisol concentration of female Malayan sun bear in BSBCC, LKW and MWC.
Malayan sun bear.The Figure3.2 displays the correlation between stress levels (cortisol concentration) and oestradiol levels in captive female Malayan sun bears.To examine the relationship between stress levels and oestradiol hormone levels, the Pearson correlation test was conducted, and the results indicate a