圈養野生動物的保種計畫中，人工繁殖技術為重要的策略應用，然先決條件需先行瞭解物種的繁殖模式，並進行長期監控。傳統採血檢測動物生殖內泌素的方法，容易對神經質的野生動物造成緊迫傷害，且影響檢測的精確性，因此利用動物自然排泄的糞尿檢測，極具開發潛力。本研究乃應用自行建立之非侵入性糞類固醇檢測技術，探討台北市立動物園在圈養環境下之台灣黑熊（Ursus thibetanus formosanus）、馬來熊（Ursus malayanus）及無尾熊 （Phascolarctos cinereus），其生殖與緊迫生理上的變化，期以長期監控動物生理狀態，提供有效的繁殖策略，透過緊迫指標的建立，作為環境良窳之評量，提升動物福利之水平。本研究共分五個部份：
Monitoring the reproductive and stress status of Formosan black bear (Ursus thibetanus formosanus)、Malayan sun bear (Ursus malayanus) and Koala (Phascolarctos cinereus) by fecal steroid
Yang Chien Jen
The purpose of the study was to use a non-invasive technique to understand the reproductive and stress physiology of bears and koala under captivity. Physiological level of crucial sex steroids and cortisol was determined to investigate the stress-induced effect on reproduction under captivity over a long period of time. Under mentioned are the investigations undertaken and their results:
Fecal samples from bears and koala were collected every two hours for three consecutive days and assayed for the level of steroids to determine the circadian rhythm over a twenty-four hours period. The results showed that progesterone (P4), testosterone (T) and cortisol (F) did not had any significant change throughout the cycle, but estrogen (E2) level was significantly high during the day in Formosan black bear. In Sun bear there was no significant change in fecal T, but P4 and F were in higher levels during the daytime. There were no obvious changes in the level of different steroids in the fecal sample of koala collected every two hours over the whole day range (9AM-3PM).
The reproductive steroid status and social behavior of Formosan black bears were investigated. The fecal samples were collected biweekly to monitor the variation of steroids level over a long period. The social behavior of Formosan black bears was observed bimonthly for a year with more frequent observations during the breeding season. Our findings revealed that fecal T level was significantly higher in breeding season in male bears and fecal P4 level in females was lower during breeding season. There was no significant change in fecal E2 level both during breeding and non-breeding seasons.
Regardless of mating status, the level of fecal P4 was found to be significantly higher during the month of October to December in similar pattern in all female Formosan black bears.
Abailable information of breeding in Taiwan revealed that the Formosan black bear conceived in March and parturated in November to December, delivering a small cub. This unique feature indicates the phenomenon of delayed implantation. Three weeks before mating there was significant increase in fecal E2, while at the period of mating there was a spike of fecal P4. However, there was no significant change in fecal P4 level, one month before and two months after mating, while fecal E2 level changed significantly a month prior to mating with no change in months that followed. It was also found that levels of P4 and E2 were significantly higher in the month of November compared to other months of the year. The social behavior gradually increased to 15~20% two weeks before mating, occupying approximately 40% of daily activity at peak during the mating period. There was no seasonal variation in the level of P4 in Sun bear, while E2 level was reflected with the changing reproductive status. The T levels of male Sun bears were influenced by the presence of females, with significant increase when they were in estrus.
The female koala returned to estrous 52 days after previous mating in non-parturient animal, with similar pattern of P4 level to that of parturient ones. It was found that level of P4, 52 days post mating remained at low level in parturient female. This valuable information can help us predict the presence of joey in the pouch of female Koala, which can provide basis for advance managerial approaches.
In spite of the changes in seasonal fecal T levels, the male Koalas were found to be a year round breeders, indicating weak reflection of fecal steroid level and existing reproductive status.
The influence of exogenous stimulation of anesthetic agent and adrenocorticotropic hormone (ACTH) on the blood and fecal F levels were investigated. Post administration of anesthetic agent, the blood steroid level increased three to five times within 40 minutes and then dropped gradually reaching normal status after two hours. When ACTH was administered immediately after it, the blood F level increased rapidly and maintained high level even after two hours. When the anesthetized bear’s fecal F level was monitored, it remained at higher level for three consecutive days, but when anesthesia was immediately followed by ACTH treatment, the level of F remained at higher level only for a day.
The influences of environmental factors to the behavioral and fecal steroid of Formosan black bear were also investigated. The switch in the place of the female Formosan black bears induced different effects on individual animals. It was observed that individual animals were under different degree of stress reflected in fecal F level.
A female Formosan black bear suspected for being pregnant was isolated for a period of four months, first month in isolation saw the level of fecal F increased by about two times. Then, there was a sharp drop in F level, even below the pre isolation level, which remained for around three months. The bear was released from isolation as no further signs of pregnancy noticed, instead estrus signs were manifested. Environment enrichment decreased the larger stereotyped behavior in male black bear while exploratory behavior was found to increase obviously. However, we found that after some time the bear regained it stereotyped behavior with adjustment to the new environment, requiring further intervene to enrich the surrounding to suppress it. There was difference in the fecal F level in male and female bears subjected to environment enrichment. The male did not show any significant change while the female had a decreased level of F.
This is the first study of its type that has employed non-invasive study over a long term on bears and koalas relating the investigation of fecal steroids and physiology. The sex steroids and F level in the feces was monitored to obtain a daily circadian rhythm, yearlong reproductive status, and changes as a result of isolation and environment enrichment. We conclude that this novel non-invasive technique will be of important help in understanding the physiological status of animals under captivity. The informations obtained by this technique can be a useful tool to enhance the health, procreation and welfare of the animals under captive condition.