應用糞便類固醇內泌素以監測森林性陸龜之緊迫與生殖

全世界現存的淡水龜及陸龜有三百多種，逾半數物種遭遇到滅絕的危機且狀況持續惡化。其中又以分布於亞洲的種類因食用、藥用或人為過度獵捕等因素，導致多達九成的亞洲龜類面臨物種存續的危機，因此必須採取域外保育與人工復育方式以確保這些物種的延續。以非侵入性的糞便採集，配合酵素免疫分析法（enzyme-linked immunosorbent assay，ELISA）來檢測其糞便中類固醇內泌素（fecal steroid hormone）是目前最常用來研究野生動物生殖與緊迫生理（reproductive and stress physiology）的方法。本研究首先利用台北市立動物園野生動物收容中心所飼養的26隻紅腿象龜（Chelonoidis carbonaria）進行不同飼養密度（rearing density）對緊迫生理影響的實驗，以分析其糞便中皮質固酮濃度（fecal corticosterone level）。結果顯示，夏季所測得的糞便皮質固酮濃度顯著高於冬季(P < 0.05)，而不同性別與不同飼養密度進行比較皆有顯著差異（P < 0.05）。高飼養密度確實導致陸龜體內的皮質固酮濃度上升，進而促使血液中異嗜球/淋巴球比值（heterophils to lymphocytes, ‘H : L’ ratios）升高。為了累積更多瀕危龜類基礎生物學的珍貴資料，本研究亦針對緬甸星龜（Geochelone platynota）糞便性類固醇內泌素（fecal sex steroid hormone）濃度變化進行監測，並配合超音波影像（ultrasonography）觀察雌性個體的生殖週期變化。結果顯示，在春季氣溫逐漸上升時，雌龜的孕酮（progesterone）與雌二醇（estradiol）濃度伴隨著體內的濾泡發育而升高，當濾泡成長至3公分左右會開始排卵（ovulation），而孕酮與雌二醇濃度也隨之下降。但每隻個體的產蛋組（clutch）與產蛋間期（interesting period）可能因體型、年齡等因素而出現很大的差異。雄性個體糞便睪固酮（fecal testosterone）濃度也在春季上升至最高峰，同時可觀察到頻繁的交配行為。綜觀上述，利用酵素免疫分析法分析糞便樣本，對於動物沒有侵入性與傷害，並能有效監測其體內的類固醇內泌素（hormone）變化，以便了解各種瀕危龜類的生殖週期情況，盼能結合域內與域外保育，有助於物種復育之進行。

關鍵字

紅腿象龜；緬甸星龜；酵素免疫分析法；皮質固酮；睪固酮；孕酮；雌二醇

並列摘要

There are about 300 living tortoise and freshwater turtle species in the world. However, half of them are threatened with extinction. In Asia, of all the threats that turtles face, the most urgent is over harvesting due to great demand of food and traditional medicine. According to recent statistics, 90% of the Asia species are facing the crisis of extinction; therefore, it is crucial to adopt ex situ conservation and artificial restoration to insure species survival. Non-invasive enzyme-linked immunosorbent assays（ELISA）for fecal steroid hormone analysis is currently the most common method used in studies of wildlife fauna reproduction and stress physiology. In this study, we evaluate the influence of rearing density on stress condition of 26 adopted red footed tortoise（Chelonoidis carbonaria）in Taipei Zoo Wildlife Rescue Center by analyzing their fecal corticosterone levels. Our results revealed that the fecal corticosterone levels were significantly higher in medium and high density groups. However, female tortoises were more sensitive to rearing density compared to male individuals. Interestingly, we found the basal corticosterone levels were varied in different seasons: feces samples collected in summer showed higher corticosterone concentrations than in winter. Further, to make repopulation of tortoise more efficient, we monitored the reproductive cycles of Burmese star tortoise（Geochelone platynota）by analyzing fecal sex steroid hormones and ultrasonography. The results revealed that as the temperature rose gradually in spring, the follicles began to grow accompanied with progesterone and estradiol concentrations increase in females. And after ovulation occurred as follicles reached the size of 3.0 cm, progesterone and estradiol concentrations went down. However, the clutch number per breeding season and inter-nesting periods were different from individual to individual as described in previous studies. On the other sides, male individuals had maximal testosterone concentrations and frequent mating behaviors in spring. In conclusion, we demonstrated that the non-invasive ELISA on fecal steroid measurement is an applicable method for monitoring stress status and reproduction cycles of tortoise. And this technique might help reproduction of endangered tortoise, integrating ex situ and in situ re-introduction programs, and species survival projects.