大熊猫粪便样本处理与激素萃取方法比较

A comparative study on fecal sample pretreatment and hormone extraction methods in giant pandas(Ailuropoda melanoleuca)

通过测定圈养动物粪便中的激素水平,监测其发情与怀孕状态和评估其福利状况是目前普遍采用的一种实验手段。然而,现有研究对于圈养野生动物粪便样本的处理及其激素萃取方法存在很大分歧,研究结果也不尽一致。本研究采集了10只(雌∶雄=1∶1)圈养大熊猫(Ailuropoda melanoleuca)的粪便样本,分别采用冷冻干燥-研筛法和冷冻干燥-粉碎法处理粪便样本,然后分别使用80%甲醇、90%乙醇和95%乙醇3种萃取剂进行激素萃取,最后比较2种粪便处理方法和3种溶剂萃取方法所获得的粪便样本中孕酮、雌二醇、睾酮和皮质醇的浓度。结果表明,冷冻干燥-研筛法结合80%甲醇溶液萃取处理的样本中雌二醇和孕酮检测值最高,冷冻干燥-粉碎法结合80%甲醇溶液萃取处理的样本中睾酮检测值最高。冷冻干燥-粉碎法结合90%乙醇溶液萃取处理的样本与冷冻干燥-研筛法结合80%甲醇溶液萃取处理的样本中皮质醇检测值更高,且2种方法的检测结果差异不显著。综合粪便处理难易程度和萃取溶剂性质,建议雌二醇、孕酮和睾酮激素检测根据最高检测浓度的方法进行粪便前处理,皮质醇激素检测使用冷冻干燥-粉碎法结合90%乙醇溶液萃取方法进行粪便前处理。本研究为动物粪便生理指标测定提供了技术指导,可应用于野生动物生理生态、繁殖生态和和保护生物学相关研究。

It is a common protocol to monitor and detect the estrus and pregnant state in captive animals and evaluate their stress level and welfare using non-invasive sampling methods and fecal hormone assay.The current methods for the treatment of fecal samples and extraction of fecal hormones in captive wild animals,however,are highly debated and the results are inconsistent in different studies.In this study,we collected fecal samples from 10 captive giant pandas(male∶female=1∶1)and used two different fecal sample treatment methods,freeze-drying sifting and freeze-drying crushing,to treat those fecal samples.Next,we used three fecal hormone extraction methods,i.e.,80%methanol,90%ethanol,and 95%ethanol extraction.Last,we compared the concentration of progesterone,estradiol,testosterone,and cortisol in each sample treated and extracted by different methods and solutions.The results show that estradiol,progesterone,and cortisol extracted by freeze-drying sifting method combined with 80%methanol solution,and testosterone extracted by freeze-drying crushing method combined with 80%methanol solution had the highest hormone content among all the treated fecal samples.The results of cortisol detection showed that the concentration of cortisol obtained by freeze-drying crushing method combined with 90%ethanol extraction and freeze-drying sifting method combined with 80%methanol solution extraction was higher,and the difference between the two methods was not significant.Considering methanol is highly toxic and difficult to obtain and the crushing method is convenient and fast to treat feces,it is suggested that the pretreatment of estradiol,progesterone,and testosterone in feces should be carried out according to the method of maximum hormone detection,and pretreatment of cortisol in feces should be carried out by freeze-drying crushing method combined with 90%ethanol extraction.This study provides technical guidance for obtaining more accurate and effective physiological indicators for captive and wild animals.It can be applied to broad taxa of wild animals and biodiversity conservation.