Effect of chronic crowding stress
Captive environment is much different than wild environment, thus it would potentially stressed captive animals. There are many stressors in captive environment , and stress effect may be existing even when animals are reared in captivity for many generations . Crowding is one of the most important stressors in captive environment, which means less space and close contact with conspecifics. Most studies reported higher glucocorticoid levels according to crowding. For example, plasma cortisol levels of non-human primate in crowding groups were significantly higher than paired individuals [13–15]. Li et al.  found that changes from large enclosures to small pens resulted in higher level of cortisol secretion in Pe’re David’s deer (Elaphurus davidianus).
However, in this study, compared with less crowding condition (217.1 ug/g), female musk deer housed in higher crowding condition (177.2 ug/g) had significantly lower FCC (Figure 1). These inconsistent glucocorticoid responses to crowding may due to different stress time, and different personality of species—different species may responses to the same stressors differently [1, 2]. Forest musk deer are territory, solitary, shy, and timid mammals in the wild [11, 17]. However, in captivity, they are compelled to be housed with each other in an enclosure that is 40 to 132m2. No doubt that the increased social contact and decreased space were stressful for such solitary and shy animals. What’s more, forest musk deer like jumping and running, that’s their survival strategies evolved in the wild. However, the captive space is too small to exhibit this behavior, and they developed stereotyped behavior (such as repeated walk back and forth, constant jump up and down). Our investigation in other breeding centers show that forest musk deer in semi-free captive environment (more natural environment, larger space that is about 660 m2 and about 200-300 m2 per deer) have significantly lower stereotyped behavior than those in captive environment (certainly, the stereotyped behaviors did not disappeared). And we found that, adults in semi-free captive environment have less disease, such as diarrhea, dyspepsia, and abscess disease (unpublished data). Liu et al.  also reported that diseases of forest musk deer decreased when they were reared in natural enclosures (about 400 m2). In this study, the rearing pattern is one male with three female musk deer, the decreased cortisol levels of musk deer might be the response to close contact with conspecifics and lack of home range, which might become chronic stress as time passed.
Miller et al.  reviewed that chronic stress will cause both increase and decrease of glucocorticoid levels. Shortly after the stress has begun, the hypothalamic-pituitary-adrenal axis may become activated, resulting in elevated corticoid output. However, with the passage of time, the body could mount a counter-regulatory response such that corticoid output rebounds below normal . Recently, many researchers found the decreased glucocorticoid levels in captive or wild animals. For example, in free-living and wild-caught European starlings (Sturnus vulgaris) exposed to an experimental chronic stress consisting of unpredictable, and different rotating stressors, both baseline and stress-induced corticosterone are suppressed [20, 21], and sensitivity of the pituitary and adrenal gland are altered . Linklater et al.  also suggested that captivity results decline of fecal corticoid levels in rhinoceros after translocation. In our study, the decreased cortisol levels of the captive forest musk deer may reflect the end stage of stress—because all of the experimental musk deer have been captive reared since they were born, such long time may bring them into the end status of the chronic stress.
An interesting but inexplicable phenomenon is that crowding seems don’t affect stress of male forest musk deer, because males in both groups had similar FCC. One possibility is that male musk deer are highly stressed under captive environment, which might cover the crowding effect. Much more has been discussed below.
Sex difference of cortisol levels in heterosexual groups
In our study, crowding environment means less space and close social contact with conspecifics. Animals cope with social contact differently according to group type, group size, and gender. Our study show an apparently different cortisol levels in different sexes, males had significantly lower FCC than females (19.9% and 54.5% higher in female than in male). Sex differences in adrenocortical activity are not uncommon in other mammals. For example, female North American clouded leopards (Neofelis nebulosa) and female mouse lemur (Microcebus murinus) had significantly higher corticoid levels than males [15, 23]. Other studies about glucocorticoid levels in rodents and humans according to gender are inconsistent. For example, adrenal weight is greater in the male hamster than in the female . However, other studies about rats showed that corticosterone concentration were higher in female than male rats [25, 26]. These sex differences may due to different gonadal hormone effect [27–30], and glucocorticoid receptor and binding protein levels [30, 31]. The sex differences may reflect underlying differences in steroid metabolism, excretion routes, and pituitary responsiveness .
However, in this study, the sex differences about cortisol levels in forest musk deer might reflect different stress status. Many studies about rodent and humans show that the female can influence male’s behavior and physiology [33, 34]. Naturally, musk deer are solitary, even the female and male musk deer encounter and stay with each other briefly only during breeding seasons, and they separate and return to their own territories as soon as mating succeeded/ended . In captivity, several male forest musk deer are reared together with several females in an enclosure (in our study, the rearing pattern is one male with three female), all the year round. Such unnatural environment may cause chronic stress; female musk deer may enhance the stress of male individuals. Our investigation show that, when the male and the female forest musk deer are reared separately during the non-breeding season—several males live together in an enclosure without see each other (everyone has an individual house), and are allowed by turns to play and move in the outside yard. Their fecal cortisol levels are similar with females at the non-breeding seasons. So it could be speculated that, the long-term exist of female musk deer might enhance the stress of males, leads to lower cortisol levels in the males. It seems to explain, at least part of, the reasons of higher mortality in the male than in the female forest musk deer (, and our unpublished data). And during the mating seasons, many centers lack the seed breeding male forest musk deer (our surveys), which might be the results of chronic captive stress. Fortunately, in recent years, some centers began semi-free rearing practices, which may improve welfare of musk deer.
However, other ungulates, such as red deer (Cervus elaphus), did not found significant differences between sexes ; but they performed the study on an undisturbed red deer herd, kept in a 45-ha enclosure. While in our study, the forest musk deer are captive in an environment with restricted space (only less than 132m2). What’s more, red deer is social species, while forest musk deer is solitary animals. In the captive environment, however, they are compelled to live with conspecifics. Such unnatural environments may induce highly stress.
It’s a pity that we did not collect wild feces of forest musk deer, and we do not know the actual stress status of forest musk deer in the wild. So the relationship of stress status of forest musk deer between the captive and wild conditions should be further studied. Furthermore, although female musk deer lived in lower crowding condition had higher cortisol levels, but we could not determine that these individuals are comfortable. It’ should be pointed out that in this study, we defined the crowding degree as relative higher and lower, because we yet do not know the threshold of un-crowding conditions.