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標題: 時間和溫度對亞洲象血液樣品中皮質醇,孕酮和睪酮穩定性的影響
Effects of time and temperature on stability of cortisol, progesterone and testosterone in blood samples of Asian elephant (Elephas maximus)
作者: 李慕瑶
Mu-Yao Li
關鍵字: 皮質醇
blood samples
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摘要: 不同物種的各種荷爾蒙的降解程度對儲存時間和溫度有不同的反應。大象非抗凝和抗凝的血液樣本在被離心前存放的溫度和時間長短對荷爾蒙的降解影響還沒有被研究過。本次實驗中設計了兩個部分: 在EDTA項目中,每只大象(Baanchang elephant camp,清邁)會被採集26 ml血液,在離心前被儲存在13管EDTA管中。其中一管被作為基礎值(T0)會被立刻離心。剩下的12管會被進一步分成三個溫度組:低溫組(4 ℃),室溫組(25 ℃)和高溫組(37 ℃),各組會被放置在相應的溫度環境內。在6小時,24小時,48小時和62小時之後,各溫度組內的一瓶樣本會被取出並離心取血漿冷凍。在血清項目中(NA),同樣採集到的另外26 ml原始血液樣本會在離心前被分裝在無抗凝劑的13管血清管內。之後對樣本的處理同在EDTA項目。所有的血漿和血清在提取之後檢測之前放在-20 ℃冷凍保存。利用酶聯免疫分析法檢測樣本中皮質醇,睪酮和孕酮的濃度。得到的原始數據會被計算成相對於T0的百分比 (% of T0)。利用One-way ANOVA和Dunnett's檢驗來分析% of T0來確定溫度,時間,溫度*時間對荷爾蒙講解對影響。顯著性差異被定為當p值小於0.05。結果:1)EDTA項目中三個溫度組內的皮質醇,睪酮和孕酮在62小時後後保持穩定;2)NA項目中高溫組的皮質醇在48小時和62小時後均出現了顯著性的下降;3)NA項目中高溫組的睪酮在48小時和62小時後出現了顯著性的下降;4)血清項目中時間和溫度都不會顯著影響孕酮的濃度。這些結果表明:1)在高溫(37 ℃)情況下,血漿樣本中各荷爾蒙有更好的穩定性;2)孕酮是檢測的三種荷爾蒙中最穩定的,可以在離心前的樣本瓶中保存至少62小時;3)建議在採樣後48小時內檢測皮質醇和睪酮。
Different hormone degradation responds differently to storage time and temperature in different species. The effects of storage time duration and temperature on hormone degradation before centrifugation have not been evaluated in elephant blood. We designed two project in this experiment. A total of 52 ml blood was collected from each elephant (Baanchang elephant camp, Chiang Mai), each project took up half of the blood. In Project EDTA, 26 ml of blood were divided into 13 aliquots and was left in EDTA tubes before centrifugation. 1 aliquot was centrifuged immediately and set as baseline (T0). 12 aliquots remained were divided into 3 temperature subgroups: group LT (4 ℃), group RT (25 ℃) and group HT (37 ℃), each group was incubated at relative temperature. One aliquot of each group was centrifuged and plasma was collected at 6hr, 24hr, 48hr, 62hr. In Project NA, 26 ml blood was divided into 13 aliquots and was left in non-anticoagulant tubes before centrifugation. All aliquots were treated like as Project EDTA. All the plasma and serum extracted was kept at -20 ℃ until analysis. Enzyme-linked immunoassay (EIA) was applied to measure the concentration of cortisol (male and female elephant), testosterone (male elephant) and progesterone (female elephant). Raw data was calculated as % of Time 0 (T0). One-way ANOVA and Dunnett's test were used to analyze % of T0, to determine the effect of time, temperature and time * temperature. Significant difference was confirmed when p < 0.05. The results were 1) cortisol, progesterone and testosterone in Project EDTA was stable at all temperature condition for up to 62 hours; 2) cortisol in Project NA decreased significantly after 48 and 62 hours in group HT (p<0.05 and p<0.01, respectively); 3) testosterone in Project NA decreased significantly after 48 and 62 hours in group HT (p<0.01 and p<0.001, respectively); 4) temperature and time duration before centrifugation did not affect concentration of progesterone in Project NA. These finding suggested that 1) plasma sample may provide better hormone stability at 37 ℃; 2) progesterone is the most stable hormone in elephant blood sample, and can be kept in tubes before centrifugation for up to 62 hours. 3) cortisol and testosterone concentration should be measured before 48 hours.
文章公開時間: 2018-12-26
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