大熊猫粪样内类固醇激素保存方法及应用研究
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摘要
目前利用非损伤性激素测定已可有效监测圈养大熊猫(Ailuropoda melanoleuca)的各项类固醇激素水平,并进一步了解其繁殖及应激状态。但对于这一方法在野生大熊猫中的应用,尚须考虑取样延迟、野外保存等多方面的问题。本文着重研究了大熊猫粪样中四种类固醇激素的常温保存时效、保存方法以及粪样的干燥方法,旨在为野生大熊猫各项非损伤性内分泌监测工作奠定一定的理论基础,并以此推动今后野生大熊猫的保护工作。研究结果如下:
1.在新鲜粪样排出后,雄性大熊猫粪样中睾酮、皮质醇以及雌性个体粪样中的睾酮,在室温条件下放置的时间不宜超过12小时;而雌性粪样中的皮质醇和雌二醇的保存时间也分别不宜超过16及24小时;孕酮则应小于8小时。
2. 95%乙醇保存法可在室温条件下稳定保存雄性皮质醇15天,雌性雌二醇45天;烘箱干燥保存法(90℃,3-4小时)可稳定保存雌二醇45天。考虑野外工作的需要,依据试验数据的稳定性,大熊猫粪样内睾酮、皮质醇、雌二醇3种激素在相同的处理和保存条件下,可在60天内进行相对值的比较研究,而孕酮则需要另行探索其它保存方式。
3.仅冷冻保存的雄性大熊猫粪样中的皮质醇及雌性个体中的雌二醇,以及使用95%乙醇常温保存粪样的孕酮经由烘箱干燥后,与冷冻干燥后的测定值间无显著性差异。因此对于冷冻保存及乙醇常温保存的粪样,均应尽可能采用冷冻干燥的方式进行处理。
4.在2007年3月-5月,北京动物园雄性大熊猫“大地”在进入发情期后,粪样中的睾酮在5月5日达到高峰值(9.06 ng/g),并在发情结束后再次下降至基础水平。而雌性个体“妞妞”在2007年未出现任何明显的发情迹象,其粪样中雌二醇水平也未出现明显的上升,总体呈无规律变化。
5.在五一黄金周期间,随着客流量的明显上升,雌雄个体粪样中的皮质醇水平均出现了明显的升高。其中雄性大熊猫粪样中的皮质醇含量的变化与前一日的客流量显著性相关,而雌性个体粪样中的皮质醇水平则与前一日客流量存在极显著的相关性。
Steroid hormone levels of the captive giant pandas (Ailuropoda melanoleuca) can be detected effectively with non-invasive methods at present, and further monitored the reproduction and stress status of them. However, many aspects should be considered to applying these non-invasive methods to the field, such as the preservation methods, the effective storage time, etc. This paper discussed the effects of different preservation methods and dry methods on four fecal steroids in giant panda, and studied the effective storage time at room temperature after defecation also, aimed at establishing certain theoretical foundation of non-invasive endocrine monitoring for wild giant panda. The main results are showed as follows:
1. Fecal testosterone and cortisol of the male giant panda, and the testosterone of female could be kept effectively for 12 hours at room temperature after defecation. The cortisol of the female could be stored for 16 hours, and estradiol for 24 hours. However, the effective storage time of fecal progesterone should be less than 8 hours.
2. Fecal cortisol of the male could be stored effectively for 15 day, and estradiol of the female for 45 days in 95% ethanol treatment; fecal estradiol of the female could be effectively stored for 45 days with oven-drying treatment (90℃, 3-4 hours) at room temperature, compared with the control samples that being lyophilized and extracted immediately. It is practically considered on basis of the data stability as well as field work, the relative values of fecal testosterone, cortisol and estradiol could be used in comparisons between groups as being preserved in the same circumstances of treatment within 60 days. The methods for storing fecal progesterone, however, address further studies.
3. The conventional oven drying method varied most of the fecal steroids significantly, except for the male cortisol and female estradiol of the frozen samples, and the female progesterone stored with 95% ethanol. When condition allowed, lyophilization method is the better way to drying both kinds of storage feces.
4. In March - May of 2007, the fecal testosterone concentrations of male giant panda "Da Di" of the Beijing Zoo increased during behavioural oestrus, and was on peaking value (9.06 ng/g) of May 5, then dropped to the basic level again after oestrus. Female individual "Niu Niu " appeared no obvious oestrus behavior in 2007, and the estradiol levels did not presented obvious rising either, and presented an irregular change.
5. With the obvious rising of tourists, the fecal cortisol concentrations increased significantly both of male and female giant panda during May Day golden week. There were significant correlation between the cortisol concentration of male and the previous-day tourists number, and very significant correlation between the cortisol concentration of female and previous-day tourists number.
1.在新鲜粪样排出后,雄性大熊猫粪样中睾酮、皮质醇以及雌性个体粪样中的睾酮,在室温条件下放置的时间不宜超过12小时;而雌性粪样中的皮质醇和雌二醇的保存时间也分别不宜超过16及24小时;孕酮则应小于8小时。
2. 95%乙醇保存法可在室温条件下稳定保存雄性皮质醇15天,雌性雌二醇45天;烘箱干燥保存法(90℃,3-4小时)可稳定保存雌二醇45天。考虑野外工作的需要,依据试验数据的稳定性,大熊猫粪样内睾酮、皮质醇、雌二醇3种激素在相同的处理和保存条件下,可在60天内进行相对值的比较研究,而孕酮则需要另行探索其它保存方式。
3.仅冷冻保存的雄性大熊猫粪样中的皮质醇及雌性个体中的雌二醇,以及使用95%乙醇常温保存粪样的孕酮经由烘箱干燥后,与冷冻干燥后的测定值间无显著性差异。因此对于冷冻保存及乙醇常温保存的粪样,均应尽可能采用冷冻干燥的方式进行处理。
4.在2007年3月-5月,北京动物园雄性大熊猫“大地”在进入发情期后,粪样中的睾酮在5月5日达到高峰值(9.06 ng/g),并在发情结束后再次下降至基础水平。而雌性个体“妞妞”在2007年未出现任何明显的发情迹象,其粪样中雌二醇水平也未出现明显的上升,总体呈无规律变化。
5.在五一黄金周期间,随着客流量的明显上升,雌雄个体粪样中的皮质醇水平均出现了明显的升高。其中雄性大熊猫粪样中的皮质醇含量的变化与前一日的客流量显著性相关,而雌性个体粪样中的皮质醇水平则与前一日客流量存在极显著的相关性。
Steroid hormone levels of the captive giant pandas (Ailuropoda melanoleuca) can be detected effectively with non-invasive methods at present, and further monitored the reproduction and stress status of them. However, many aspects should be considered to applying these non-invasive methods to the field, such as the preservation methods, the effective storage time, etc. This paper discussed the effects of different preservation methods and dry methods on four fecal steroids in giant panda, and studied the effective storage time at room temperature after defecation also, aimed at establishing certain theoretical foundation of non-invasive endocrine monitoring for wild giant panda. The main results are showed as follows:
1. Fecal testosterone and cortisol of the male giant panda, and the testosterone of female could be kept effectively for 12 hours at room temperature after defecation. The cortisol of the female could be stored for 16 hours, and estradiol for 24 hours. However, the effective storage time of fecal progesterone should be less than 8 hours.
2. Fecal cortisol of the male could be stored effectively for 15 day, and estradiol of the female for 45 days in 95% ethanol treatment; fecal estradiol of the female could be effectively stored for 45 days with oven-drying treatment (90℃, 3-4 hours) at room temperature, compared with the control samples that being lyophilized and extracted immediately. It is practically considered on basis of the data stability as well as field work, the relative values of fecal testosterone, cortisol and estradiol could be used in comparisons between groups as being preserved in the same circumstances of treatment within 60 days. The methods for storing fecal progesterone, however, address further studies.
3. The conventional oven drying method varied most of the fecal steroids significantly, except for the male cortisol and female estradiol of the frozen samples, and the female progesterone stored with 95% ethanol. When condition allowed, lyophilization method is the better way to drying both kinds of storage feces.
4. In March - May of 2007, the fecal testosterone concentrations of male giant panda "Da Di" of the Beijing Zoo increased during behavioural oestrus, and was on peaking value (9.06 ng/g) of May 5, then dropped to the basic level again after oestrus. Female individual "Niu Niu " appeared no obvious oestrus behavior in 2007, and the estradiol levels did not presented obvious rising either, and presented an irregular change.
5. With the obvious rising of tourists, the fecal cortisol concentrations increased significantly both of male and female giant panda during May Day golden week. There were significant correlation between the cortisol concentration of male and the previous-day tourists number, and very significant correlation between the cortisol concentration of female and previous-day tourists number.
引文
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