滤膜过滤法在大熊猫尿液采集及代谢组学中的应用

A Study on the Usage of Filtration Membrane Urine Collection and Application in Metabolisms in Ailuropoda melanoleuca

圈养动物尿液中含有丰富的代谢产物,是代谢组学分析中用以判定动物发情、妊娠、健康状态、应激水平及动物福利的重要生物检材。因采样困难及安全考虑,目前多选择动物离开或转移后采集遗留在圈舍地面的尿液样本,这种样本容易被污染而影响检测结果,因此探索一种低污染、方便且快捷的尿液采集和预处理方法非常必要。针对8只(3♀,5♂)圈养大熊猫(Ailuropoda melanoleuca)的27份尿液样品,分别采用不做处理(对照组)、0.22μm滤膜过滤和添加叠氮钠3种预处理方法,然后通过微生物培养、核磁共振分析及代谢物推定,检验并评估了滤膜处理样品的效果。结果发现:经过滤膜处理的尿液样品培养后无细菌,3个处理组间代谢物的差异不显著;进一步的分析还发现,不同性别及年龄组大熊猫的尿液中分别发现7种和8种差异性代谢产物(p<0.05)。滤膜过滤法可作为大熊猫尿液标准化采样方法用于代谢组学分析,该结果不仅为大熊猫健康与发情状态监测和应激水平判断提供了重要支撑,也可以推广应用到其他圈养野生动物的尿液采集及代谢组学分析。

Captive animal urine is rich in metabolites which is an important biological material for determining its estrus,pregnancy,health status,stress level and animal welfare by means of metabolite technique.However,urine samples are difficult to collect directly from the animal due to safety considerations.Urine samples were usually collected from the floor of animal’s enclosure after the animal’s excretion and moved to other place of the enclosure.Those samples are often con⁃taminated by bacteria,which in turn affect the results of metabolite determination.It is necessary to explore a low-polluting and convenient urine collection and pretreatment method.In this paper,we collected 27 urine samples from eight captive gi⁃ant pandas(Ailuropoda melanoleuca)(3♀,5♂)by traditional methods,then each sample was divided into three groups for the control,filtrating with 0.22μm filtration membrane and adding sodium azide treatments,respectively.Next,we used mi⁃crobial cultures and Nuclear Magnetic Resonance(NMR)analysis and metabolite analysis to assay the bacteria removal effi⁃cacy of three treatments.The results showed that no bacteria were cultured in urine samples treated by filtration membrane,and there were no significant differences in the metabolites of NMR among three treatments.Further analysis found that seven and eight urinary metabolites significantly differed in genders and differed among age groups(both p<0.05),respec⁃tively.The results suggest that the filter membrane pretreatment is effective for removing bacteria from urine samples,and can be used as a standard method for urine sample pretreatment and further evaluation of physiological indexes of giant pan⁃das in metabolomics.Our findings not only enrich the basic metabolism database of giant pandas,and also provide data support for the health status monitoring,estrus and stress level assay in giant pandas and other captive wild animals.