**4. Discussion**

Lemurs in captivity are exposed daily to potentially stressful situations. This is even more pronounced in the case of temporary housing facilities. In addition to the normal daily routine, which includes regular morning cleaning of all quarters and providing food twice a day, construction work was in progress in the monitored facility. Moreover, the observed lemurs were moved, and new animals arrived during the observation period. Such activities that are not carried out regularly are especially likely to cause stress to animals. In all lemur species, significantly increased FGM levels were found during the monitored period. The fluctuation of FGM levels could be caused by the recorded stressors, as elevated concentrations were often found 1 to 2 days after the events that deviated from the daily routine. While blood glucocorticoid concentrations increased shortly after exposure to an acute stressor, and in faeces, the increase of glucocorticoids resulting from stress was seen with a delay [7].

On the 13th and 14th day of the monitored period, all-day construction work was carried out near the room where the ring-tailed lemurs were housed. In the days after (14th and 15th day) a significant increase (*p* < 0.05) in the mean FGM levels was found. This increase could result from ongoing construction work as lemurs were exposed to increased noise and vibrations in their cages. The mean FGM levels also increased on day 17 (*p* < 0.01) and day 24 (*p* < 0.05). These increases could be related to relocations of two females with o ffspring (R3 and R4) into separate cages (16th and 23rd day). The separation was necessary due to agonistic behaviour observed in the females. Despite the females being familiar with each other from their original location, stress from the new environment could cause changes in their behaviour. Relocation can be perceived as a stressor in captive wild animals because they perceive all changes and new situations as a potential threat [28]. On day 23, one female (R1) was moved to a separate cage in the same room due to an injury. The injured female exhibited no significant increase in FGM levels, however, mean FGM levels in the group of ring-tailed lemurs increased. Finally, also on the last day of monitoring, a significant increase in the mean FGM values (*p* < 0.01) was observed. According to records, two giant otters (*Pteronura brasiliensis*) were relocated into the facility on the previous day which could be stressful for lemurs housed nearby. In the monitored period, relatively stable FGM levels were found on days which were not preceded by any exceptional events (daily routine was followed). Assumedly, the above-mentioned disturbances were responsible for the changes in FGM levels and they showed a consistent one-day delay before elevating FGM concentrations. White-headed lemurs were exposed to the same potentially stressful events as the ring-tailed lemurs. Construction work was in progress on day 13 and 14 near their enclosure, which could explain a significant increase (*p* < 0.05) in the mean FGM levels on days 15 and 16. Interestingly, there was a two-day delay before the FGMs elevated in white-headed lemurs in contrast with the one-day delay in ring-tailed lemurs following the same stressor. The significantly elevated levels of FGMs were found also in the last days of monitoring (day 26 to 29). The reason is unclear. The records did not show any deviations from the daily routine. However, the animals might have responded even to some minor changes not detected or perceived by the caretakers. According to Morgan and Tromborg [2], animals can respond to the inability to escape from human reach and the daily routine of cleaning the enclosure. Although routine is a repeated activity for a human being, an animal in captivity may perceive this situation di fferently and may be stressed. For example, primates have been shown to have high heart rates at the time of the keeper's arrival at the enclosure due to routine cleaning [2]. Thus, for some more sensitive individuals, the presence of a person in their vicinity can be stressful, especially if they are in an unknown place [2,29].

In both, the ring-tailed lemurs and the white-headed lemurs, a significant (*p* < 0.05) increase in FGM levels was recorded following the construction work throughout the day in the yard of the facility where both species were housed. As Morgan and Tromborg [2] reported, excessive noise caused by, for example, noisy construction work, is stressful and a ffects not only the animal's behaviour but also the levels of specific stress markers. In captive wild animals, acute and chronic stress caused by di fficulties in coping with stressors such as public presence and noise, among others, can induce a significant increase in FGMs and repetitive pathological behaviours such as stereotypies [30,31].

Collared brown lemurs were housed in a di fferent room than the other two lemur species. Thus, they could be a ffected by di fferent potentially stressful events. In particular, these included necessary relocations due to the occurrence of agonistic behaviour in the group. In the monitored period, a significant increase (*p* < 0.05) in the mean FGM levels was found on days 17, 18, 20, 24 and 30, which could be caused by aggression and subsequent relocations. Mutual fights escalated especially during feeding. Aggressive behaviour has been proven to a ffect FGM levels, with dominant individuals having higher glucocorticoid levels [32–34]. However, also the movement itself or other changes in the group a ffect the level of the FGMs, as was demonstrated in the ring-tailed lemurs.

While the significant increase of FGMs in ring-tailed and white-headed lemurs was caused the next day after the arrival of giant otters, in collared brown lemurs no significant change was found in FGM levels following the arrival of Bennett's wallabies (*Macropus rufogriseus*) to the same room. Furthermore, in collared brown lemurs there was no increase in the mean FGM levels in response to the construction work carried out on the 13th and 14th day of the observation, likely because the collared brown lemurs were located on the other side of the facility, thus no noises or vibrations from the construction work were recorded here.

During the monitored period, handling of animals was most often associated with their relocation to other cages due to agonistic behaviour. For relocation, capture and direct handling was necessary. That can be perceived as a strong stressor [24,28] with handling duration a ffecting the stress response and subsequently FGM levels [35]. However, it also depends on other factors, such as temperament of the individual and previous experience with the stressor. Considering their activity, it is generally recommended to capture lemurs in early morning hours and only by experienced caretakers [36,37]. The lemurs who were relocated in our study were captured by an experienced caretaker with the use of net and transferred to the new cage. Capture of animals is considered an acute stressor, but it can have longer lasting e ffects if it is carried out repeatedly in a short period [38]. According to Balcombe et al. [38], handling of animals is one of the most common husbandry practices but it results in variously strong stress responses and a ffects the activity of the immune system of animals. Hämäläinen et al. [24] measured the level of stress in grey mouse lemurs (*Microcebus murinus*) based on the determination of elevated levels of FGMs after capture. Although elevated levels have been recorded in some cases, routine capture has not had long-term consequences. The stress response in black howler monkeys (*Alouatta caraya*) was evaluated by Rangel-Negrín et al. [39]. The results showed that after capture, the levels of FGMs were a ffected. Similarly, Volfova et al. [18] recorded the increase of FGMs in response to handling in ring-tailed lemurs. Captive wild animals are expected to have a stronger response to handling stress than domesticated animals [40]. Given that handling is one of the primary stressors for captive wild animals, it is essential to monitor its potential negative impact on their welfare and avoid it as much as possible.

The results show that all the three monitored lemur species respond during the observed period to specific stressful situations by increasing (*p* < 0.05) the FGM levels similarly within one to two days after the event. This delay from the stress event to GC excretion into faeces was confirmed not only in previous studies regarding handling of the ring-tailed lemurs and transportation of the black-and-white ru ffed lemurs, but also in many other studies concerning primates [13,21,23,24,41]. Generally, the delay is 24 to 48 h in primates [22]. In white-headed lemurs, however, di fferences in response to potentially stressful situations were reported compared to ring-tailed lemurs. Increased average levels of the FGMs were found more frequently in white-headed lemurs than in ring-tailed lemurs. The results may indicate that this species responds more sensitively to changes in its surroundings. Nevertheless, these di fferences may also be due to the di fferent temperament of the animals, as some studies confirm [6,7], as well as by the di fferences in the structure of the gastrointestinal tract in di fferent lemurs species [7,42], which a ffects the amount of glucocorticoids secreted into the faeces [6]. It has been proven that there are di fferences in the metabolism of glucocorticoid hormones and their secretion into faeces between di fferent species [6,43]. However, closely related species can be expected to have a similar or even equal representation of glucocorticoid hormone metabolites in their faeces and the time for which there is a demonstrable increase in the FGMs.

The differences in the FGM concentrations in the individual species may be also due to different proportions of sex in the observed groups [44], because sex has a demonstrable impact on glucocorticoid levels, with females in oestrous, pregnan<sup>t</sup> or lactating exhibiting elevated circulating glucocorticoids [6]. In our case, the ring-tailed lemur group consisted of eight females and one male, which is the standard group structure even in the wild. Three of these females had offspring and were lactating; the GC levels measured in their faeces thus increased the mean FGMs of the whole group. Similarly, Starling et al. [45] found elevated levels of the FGMs in lactating ring-tailed lemur females living in the wild. The group of white-headed lemurs, where the mean FGM levels were the lowest, consisted of two males. The group of collared brown lemurs consisted of four females and two males. The influence of sex on FGM levels was also confirmed by Arias et al. [46], who reported higher cortisol levels in female lama guanicoe (*Guanaco*) compared to males due to pregnancy. Dantzer et al. [47] found female American red squirrel (*Tamiasciurus hudsonicus*) also have elevated levels of FGMs during pregnancy. Furthermore, Carrera et al. [48] found higher glucocorticoid levels to be associated with low rank (compared to high rank) and first-time mothers (compared to multiparous mothers) in geladas (*Theropithecus gelada*).

The study has shown that some situations and activities related to captive housing can result in elevated levels of FGMs in lemurs. A similar pattern of changes in the FGM levels in response to potentially stressful situations was observed in the monitored lemurs. All three lemur species in our case showed a significant increase (*p* < 0.05) in the FGM levels within one, or at most two days after the stressor exposure. Total concentrations of the FGMs differed between the species, with the highest in ring-tailed lemurs, lower in collared brown lemurs and the lowest in white-headed lemurs. In white-headed lemurs, much lower FGM levels were found in comparison with the other two lemur species, which could be interpreted as a species difference. However, it is also possible that their low levels of FGMs corresponded to the fact that as the only one their group was not divided during the monitored period, and the males were used to each other. For social species, their mutual dependence is high [49]. Furthermore, it was also the only male group. The absence of females could also lead to an overall lower level of FGMs. In contrast, the highest glucocorticoid concentrations in ring-tailed lemurs could have been caused by the presence of lactating females in the group. Furthermore, the individual animal's temperament or social status may affect the absolute FGM levels.

Limitations of the study included lack of baseline data, varying time of arrival of individual animals to the facility (and thus varying period of acclimatization or lack thereof before the commencement of the study), different number of animals and varying sex ratio in the monitored groups. However, it was not possible to experimentally design and control the structure of the monitored groups and the conditions of observation.
