野化梅花鹿冬季食物组成和健康状况的评估
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摘要
我国野生东北梅花鹿(Cervus nippon hortulorum)的数量很少,分布区域狭窄。在黑龙江省林区,还生活着一些野化梅花鹿,这些野化梅花鹿种群都是未经过野化训练就被释放或逃逸到野外的圈养东北梅花鹿的后裔。北方地区的冬季漫长且寒冷,食物资源缺乏,是动物生存最艰难的时期。2007～2011年冬季,用非损伤取样法对野生、野化和圈养梅花鹿的取食、营养、应激和免疫等内容进行了研究,为建立野外鹿类动物健康状况的评价和监测体系提供参考。
本研究对梅花鹿冬季雪地活动的痕迹特征、雌雄个体踪迹的识别方法和同域分布的有蹄类动物足迹特点进行了详细研究。用粪便显微分析法研究了生活在中俄边境处的野生梅花鹿、黑龙江通河县龙口林场和拜泉县国富林场野化梅花鹿冬季的食物组成。野生梅花鹿冬季取食17科25属35种植物,其中木本植物15科23属,草本植物2科2属。可辨认植物占食物组成比例的87.7%,其中包括81.4%的木本植物枝叶和6.3%的蒿草。生活在林区的野化梅花鹿冬季取食18科25属34种植物,其中木本植物12科19属,草本植物6科6属。可辨认植物占食物组成比例的89.5%,包括75.3%的木本植物枝叶和14.2%的草本植物。生活在农林交错区的野化梅花鹿冬季取食20科30属41种植物,其中木本植物16科21属,草本植物4科9属,包括两种作物；在不同年份和月份,野化梅花鹿取食的植物种类和比例有所差异；冬季对水的摄入主要靠采食积雪。
用目标动物取样法对野化梅花鹿的采食行为进行观察,其采食过程中低头(含与头部平行位置)采食的时间占总采食时间的87.7%,抬头采食占10.2%,仰头和卧息时的采食时间占总采食时间的2.1%。
对野化梅花鹿雪尿中的DNA进行提取和扩增,使用两组引物进行性别鉴定,结果对17个雪尿样本中的16个进行了成功鉴定,同时与野外踪迹识别判断性别的方法进行了对比。与粪便样本相比,尿液样本中的DNA更容易被提取和扩增；梅花鹿尿样中的核DNA在-20℃条件下可以被保存至少14个月而不发生严重降解；雪尿中的DNA资源还可以应用于其他基于线粒体DNA的遗传学研究。
用酶联免疫法对梅花鹿粪尿中的类固醇激素和免疫球蛋白进行了测定。不同地区、不同梅花鹿种群的性激素水平有所差异。12月初,在同一环境下,圈养和野化雄性梅花鹿粪便中睾酮的浓度都显著高于雌鹿；雌雄鹿粪便中雌二醇和孕酮的浓度差异均不显著；成年梅花鹿粪便中孕酮的浓度显著高于幼年。2月初,圈养成年雄鹿尿液中的睾酮浓度显著高于雌鹿,成年雌鹿尿液中的雌二醇浓度显著高于雄鹿。对比了不同性别、圈养和野化、不同地区和不同月份梅花鹿粪尿中皮质醇激素、IgG和IgA浓度的变化；尿液中皮质醇激素、IgG和IgA浓度的离散程度较粪便中高,反映的是动物瞬时的应激和免疫生理水平；粪尿中的皮质醇激素浓度受到营养和降雪等因素的影响；粪便中的皮质醇激素浓度与IgG、IgA的浓度无相关性,尿液中的皮质醇激素浓度与IgG、IgA的浓度相关性显著。
研究地区圈养梅花鹿的精饲料干物质中粗蛋白含量在8.9%-14.8%,粗饲料干物质中粗蛋白含量在4.3%-7.2%,日粮可以满足其越冬的最低维持需要；对野化梅花鹿采食的部分植物的营养成分进行分析,其干物质中粗蛋白含量在4.7%-13.6%,野化梅花鹿可以依实际情况自行调整其食物组成。梅花鹿粪便中的干物质含量因其采食的饲料和消化能力不同而有所差异,在26.4%-42.1%；粪氮含量占干物质的1.38%-2.17%,不能单独作为其食物营养价值的指示物。梅花鹿尿液中尿素氮与肌酐比值反映出不同地区动物个体营养状况的差异,结果显示幼体和成年雄性个体的营养状况在冬季较成年雌鹿更差；但梅花鹿在冬季营养代谢中出现负平衡和体重减轻是一种适应环境的正常生理现象,用雪尿中尿素氮与肌酐比值将动物的营养状况定为营养不良时要特别谨慎,梅花鹿营养状况正常和早期缺乏的尿素氮与肌酐比临界值可能在6-10之间。
用高效液相色谱法测定了梅花鹿尿液中嘌呤衍生物的含量,结果为幼鹿尿液中尿囊素比肌酐和嘌呤衍生物之和比肌酐极显著地高于成鹿；梅花鹿尿液中尿囊素占嘌呤衍生物总和的比例在74%-89%、尿酸为2%-10%、黄嘌呤与次黄嘌呤的和为8%-16%；尿囊素比肌酐与嘌呤衍生物之和比肌酐呈极显著相关。
随机对110份圈养和野化梅花鹿粪便样本进行寄生虫检查,只有1例圈养样本检出球虫虫卵,且数量较少,认为梅花鹿具有较强的抗寄生虫能力。
通过综合评估,认为生活在拜泉的野化梅花鹿营养状况良好,其应激和免疫水平都处于正常的生理范围之内,野化种群中的大部分个体都处于健康状态。拜泉地区的纬度较高,冬季寒冷,且处于农林交错区,生存环境比较恶劣,人为活动对其影响也较大,但梅花鹿依然显示了其较强的适应能力。
我国的野生鹿类绝大多数属于保护动物,在其生存最为艰难的冬季,可以用粪尿中的生理、生化指标并结合寄生虫检查等非损伤取样方法评估其健康状况。
The number of wild sika deer (Cervus nippon hortulorum) is quite few, the distributed region is quite narrow in the northeast of China. But there still lives some reintroduced and introduced sika deer in the forestry region of Heilongjiang province, these feral populations are the descendants of captive sika deer in Northeast of China which are released or escaped to this field before training. The winter of northern area is long and cold, lacking food resources, and it is the most difficult time for the animal to survive. In order to provides the reference for establishing the assessment of the feral sika deers'health condition and the monitor system, the non-invasive sampling method was used to investigate the ingestion, nutrition, stress and immune of the wild, feral and captive sika deer in the winter of2007-2011.
In the present study, the trace character, the identification of the male and female individuals traces, the footprint character in sympatric ungulates in winter snow field were used for investigation. The microscopic analysis was used to detect the winter food composition of the wild and feral sika deer living in Sino-Russian Border, Longkou forestry centre(Tonghe county in Heilongjiang Province),and Guofu forestry centre(Baiquan county in Heilongjiang Province). The wild sika deer takes17branches,25categories,35species of plants including xylophyte15branches,23categories, herbaceous plant2branches,2categories as food in winter. The proportion of recognizable plants in food composition was87.7%, including81.4%branches and leaves from xylophyte,6.3%wormwood grasses. The feral sika deer living in forest takes18branches,25categories,34species of plants as food in winter, which consists of xylophyte12branches,19categories, herbaceous plant6branches,6categories.89.5%of the composition of food could be recognized, including75.3%of woody plant foliage and14.2%of the herb. The feral sika deer living in the ecotone of farming and forestry takes20branches,30categories,41species of plants as food in winter, which consists of xylophyte16branches,21categories, herbaceous plant4branches,9categories, including2species. In different years and months, the species and the proportion of plants grazed by the feral sika deer were different. The ingestion of water mainly depends on eating snow in winter.
We observed the ingesting behavior of the feral sika deer by focal animal sampling, the time of bowing the head(including the parallel position with head) in ingesting process, accounting for87.7%of the total ingesting time, while the time of raising the head accounting for10.2%, looking up and lying in ingestion accounting for2.1%.
The sika deer urine ice was used for extracting and amplifying the DNA, and2groups of primer were applied to identify the gender, as a result,16in17urine ice samples were identified successfully. Compared with the fecal samples, the DNA in urine samples was easier to be extracted and amplified. The nuclear DNA in urine samples of sika deer can be kept at least14months at-20℃. The DNA resources in urine ice may also apply to other genetics researches based on the line plastochondria DNA.
The steroid hormone and immune globulin in sika deer feces and urine were detected by ELISA method. The sex hormone of sika deer population varies in different district and species. At the beginning of December, in the same circumstance, the testosterone concentration of both the captive and the feral male sika deer were obviously higher than the female ones. The concentrate diversity of estradiol and progesterone in male and female sika deer was not remarkable. The fecal progesterone concentration in the mature sika deer is obviously higher than the infancy. At the beginning of February, the testosterone concentration in the urine from captive male sika deer was variously higher than the females, while the urine estradiol concentration in the grown-up female deer was obviously higher than the male ones. After comparing with sika deers' cortisol hormone, IgG and IgA concentration change in the different gender, different district, different month, the captive and feral, we found the disperse level of concentration of uric cortisol hormone, IgG and the IgA was higher than in the feces, reflecting the animal instantaneous stress and the immunity physiology level. The fecal and uric concentration of cortisol is effected by factors such as nutrition and snowfall, etc. The fecal concentration of cortisol has no relevance with the concentration of IgG and IgA, the uric concentration of cortisol was obviosly relative to the concentration of IgG and IgA. The feral sika deer feces and urine hormone cortisol, IgG and IgA concentrations'difference was not significantly in male nor female animals.
The crude protein content in dry matter of captive sika deer concentrated feed is8.9%～14.8%, the crude protein content of coarse fodder is4.3%～7.2%, which could meet the lowest maintenance needs to survive in winter. After analysing the nutrition constituent of the partial plants eating by feral sika deer, the crude protein content was4.7%～13.6%, the feral sika deer can self-adjust its food composition based on the practical condition. About26.4%～42.1%of the dry matter content in the feces of sika deer was different depend on the different feed and digestion ability. The fecal nitrogen content account the dry matter for1.38%～2.17%, which could not be used as the referent of food nutrition value independently. In the urine of sika deer, the ratio of urea nitrogen and the creatinine reflects the different animals' nutrition condition in different areas individually, the results revealed that the nutrition condition of the infant deer and the male mature ones were worse than the female mature ones in winter. But the negative balance and the body weight reduction in winter nutrition metabolism was a kind of normal physiological phenomenon to adapt the environment, when the ratio of urea nitrogen and the creatinine were used to determine the animal nutrition condition into undernourishment, we should be more careful, because the critical value of the normal nutrition condition of sika deer and the early-phase lacking the value of urea nitrogen and the creatinine may be6-10.
The purine derivative content in the urine of sika deer was detected by high performance liquid chromatography, the result showed that the ratio of allantoin compared with creatinine and purine derivatives compared with the creatinine in the urine of fawn is obviously higher than the mature sika deer, the proportion of Allantoin accounting for the total purine derivatives in urine was74%-89%, uric acid was2%-10%, the xanthine and hypoxanthic was8%～16%, the ratio of allantoin compared with creatinine and purine derivatives compared with the creatinine presents significant correlation.
After inspecting the parasite from110captive and feral sika deer fecal samples randomly, there was only one captive sample checked out the ball insects egg, and the quantity was quiet small, therefore we considered that sika deer has the strong anti-parasite ability.
Through the synthetical assessment, we figured that the nutrition condition of the feral sika deer living in Baiquan was pretty well, its stress and the immunity level were both in the normal range, most individuals in the feral population were healthy. The latitude of Baiquan area is high, the winter is cold, which is in the mosaic of farmland and forest, the living environment is disgusting, and the artificial activity is quite rampant, fortunately, a strong adaptiveness were showed by the sika deer in our study.
Most of the wild deer are protectional aimal in China, non-invasive method such as the fecal and uric physiology, the biochemistry target and unifies the parasite inspection etc. were useful and safety to assess its state of health in cold winter.
本研究对梅花鹿冬季雪地活动的痕迹特征、雌雄个体踪迹的识别方法和同域分布的有蹄类动物足迹特点进行了详细研究。用粪便显微分析法研究了生活在中俄边境处的野生梅花鹿、黑龙江通河县龙口林场和拜泉县国富林场野化梅花鹿冬季的食物组成。野生梅花鹿冬季取食17科25属35种植物,其中木本植物15科23属,草本植物2科2属。可辨认植物占食物组成比例的87.7%,其中包括81.4%的木本植物枝叶和6.3%的蒿草。生活在林区的野化梅花鹿冬季取食18科25属34种植物,其中木本植物12科19属,草本植物6科6属。可辨认植物占食物组成比例的89.5%,包括75.3%的木本植物枝叶和14.2%的草本植物。生活在农林交错区的野化梅花鹿冬季取食20科30属41种植物,其中木本植物16科21属,草本植物4科9属,包括两种作物；在不同年份和月份,野化梅花鹿取食的植物种类和比例有所差异；冬季对水的摄入主要靠采食积雪。
用目标动物取样法对野化梅花鹿的采食行为进行观察,其采食过程中低头(含与头部平行位置)采食的时间占总采食时间的87.7%,抬头采食占10.2%,仰头和卧息时的采食时间占总采食时间的2.1%。
对野化梅花鹿雪尿中的DNA进行提取和扩增,使用两组引物进行性别鉴定,结果对17个雪尿样本中的16个进行了成功鉴定,同时与野外踪迹识别判断性别的方法进行了对比。与粪便样本相比,尿液样本中的DNA更容易被提取和扩增；梅花鹿尿样中的核DNA在-20℃条件下可以被保存至少14个月而不发生严重降解；雪尿中的DNA资源还可以应用于其他基于线粒体DNA的遗传学研究。
用酶联免疫法对梅花鹿粪尿中的类固醇激素和免疫球蛋白进行了测定。不同地区、不同梅花鹿种群的性激素水平有所差异。12月初,在同一环境下,圈养和野化雄性梅花鹿粪便中睾酮的浓度都显著高于雌鹿；雌雄鹿粪便中雌二醇和孕酮的浓度差异均不显著；成年梅花鹿粪便中孕酮的浓度显著高于幼年。2月初,圈养成年雄鹿尿液中的睾酮浓度显著高于雌鹿,成年雌鹿尿液中的雌二醇浓度显著高于雄鹿。对比了不同性别、圈养和野化、不同地区和不同月份梅花鹿粪尿中皮质醇激素、IgG和IgA浓度的变化；尿液中皮质醇激素、IgG和IgA浓度的离散程度较粪便中高,反映的是动物瞬时的应激和免疫生理水平；粪尿中的皮质醇激素浓度受到营养和降雪等因素的影响；粪便中的皮质醇激素浓度与IgG、IgA的浓度无相关性,尿液中的皮质醇激素浓度与IgG、IgA的浓度相关性显著。
研究地区圈养梅花鹿的精饲料干物质中粗蛋白含量在8.9%-14.8%,粗饲料干物质中粗蛋白含量在4.3%-7.2%,日粮可以满足其越冬的最低维持需要；对野化梅花鹿采食的部分植物的营养成分进行分析,其干物质中粗蛋白含量在4.7%-13.6%,野化梅花鹿可以依实际情况自行调整其食物组成。梅花鹿粪便中的干物质含量因其采食的饲料和消化能力不同而有所差异,在26.4%-42.1%；粪氮含量占干物质的1.38%-2.17%,不能单独作为其食物营养价值的指示物。梅花鹿尿液中尿素氮与肌酐比值反映出不同地区动物个体营养状况的差异,结果显示幼体和成年雄性个体的营养状况在冬季较成年雌鹿更差；但梅花鹿在冬季营养代谢中出现负平衡和体重减轻是一种适应环境的正常生理现象,用雪尿中尿素氮与肌酐比值将动物的营养状况定为营养不良时要特别谨慎,梅花鹿营养状况正常和早期缺乏的尿素氮与肌酐比临界值可能在6-10之间。
用高效液相色谱法测定了梅花鹿尿液中嘌呤衍生物的含量,结果为幼鹿尿液中尿囊素比肌酐和嘌呤衍生物之和比肌酐极显著地高于成鹿；梅花鹿尿液中尿囊素占嘌呤衍生物总和的比例在74%-89%、尿酸为2%-10%、黄嘌呤与次黄嘌呤的和为8%-16%；尿囊素比肌酐与嘌呤衍生物之和比肌酐呈极显著相关。
随机对110份圈养和野化梅花鹿粪便样本进行寄生虫检查,只有1例圈养样本检出球虫虫卵,且数量较少,认为梅花鹿具有较强的抗寄生虫能力。
通过综合评估,认为生活在拜泉的野化梅花鹿营养状况良好,其应激和免疫水平都处于正常的生理范围之内,野化种群中的大部分个体都处于健康状态。拜泉地区的纬度较高,冬季寒冷,且处于农林交错区,生存环境比较恶劣,人为活动对其影响也较大,但梅花鹿依然显示了其较强的适应能力。
我国的野生鹿类绝大多数属于保护动物,在其生存最为艰难的冬季,可以用粪尿中的生理、生化指标并结合寄生虫检查等非损伤取样方法评估其健康状况。
The number of wild sika deer (Cervus nippon hortulorum) is quite few, the distributed region is quite narrow in the northeast of China. But there still lives some reintroduced and introduced sika deer in the forestry region of Heilongjiang province, these feral populations are the descendants of captive sika deer in Northeast of China which are released or escaped to this field before training. The winter of northern area is long and cold, lacking food resources, and it is the most difficult time for the animal to survive. In order to provides the reference for establishing the assessment of the feral sika deers'health condition and the monitor system, the non-invasive sampling method was used to investigate the ingestion, nutrition, stress and immune of the wild, feral and captive sika deer in the winter of2007-2011.
In the present study, the trace character, the identification of the male and female individuals traces, the footprint character in sympatric ungulates in winter snow field were used for investigation. The microscopic analysis was used to detect the winter food composition of the wild and feral sika deer living in Sino-Russian Border, Longkou forestry centre(Tonghe county in Heilongjiang Province),and Guofu forestry centre(Baiquan county in Heilongjiang Province). The wild sika deer takes17branches,25categories,35species of plants including xylophyte15branches,23categories, herbaceous plant2branches,2categories as food in winter. The proportion of recognizable plants in food composition was87.7%, including81.4%branches and leaves from xylophyte,6.3%wormwood grasses. The feral sika deer living in forest takes18branches,25categories,34species of plants as food in winter, which consists of xylophyte12branches,19categories, herbaceous plant6branches,6categories.89.5%of the composition of food could be recognized, including75.3%of woody plant foliage and14.2%of the herb. The feral sika deer living in the ecotone of farming and forestry takes20branches,30categories,41species of plants as food in winter, which consists of xylophyte16branches,21categories, herbaceous plant4branches,9categories, including2species. In different years and months, the species and the proportion of plants grazed by the feral sika deer were different. The ingestion of water mainly depends on eating snow in winter.
We observed the ingesting behavior of the feral sika deer by focal animal sampling, the time of bowing the head(including the parallel position with head) in ingesting process, accounting for87.7%of the total ingesting time, while the time of raising the head accounting for10.2%, looking up and lying in ingestion accounting for2.1%.
The sika deer urine ice was used for extracting and amplifying the DNA, and2groups of primer were applied to identify the gender, as a result,16in17urine ice samples were identified successfully. Compared with the fecal samples, the DNA in urine samples was easier to be extracted and amplified. The nuclear DNA in urine samples of sika deer can be kept at least14months at-20℃. The DNA resources in urine ice may also apply to other genetics researches based on the line plastochondria DNA.
The steroid hormone and immune globulin in sika deer feces and urine were detected by ELISA method. The sex hormone of sika deer population varies in different district and species. At the beginning of December, in the same circumstance, the testosterone concentration of both the captive and the feral male sika deer were obviously higher than the female ones. The concentrate diversity of estradiol and progesterone in male and female sika deer was not remarkable. The fecal progesterone concentration in the mature sika deer is obviously higher than the infancy. At the beginning of February, the testosterone concentration in the urine from captive male sika deer was variously higher than the females, while the urine estradiol concentration in the grown-up female deer was obviously higher than the male ones. After comparing with sika deers' cortisol hormone, IgG and IgA concentration change in the different gender, different district, different month, the captive and feral, we found the disperse level of concentration of uric cortisol hormone, IgG and the IgA was higher than in the feces, reflecting the animal instantaneous stress and the immunity physiology level. The fecal and uric concentration of cortisol is effected by factors such as nutrition and snowfall, etc. The fecal concentration of cortisol has no relevance with the concentration of IgG and IgA, the uric concentration of cortisol was obviosly relative to the concentration of IgG and IgA. The feral sika deer feces and urine hormone cortisol, IgG and IgA concentrations'difference was not significantly in male nor female animals.
The crude protein content in dry matter of captive sika deer concentrated feed is8.9%～14.8%, the crude protein content of coarse fodder is4.3%～7.2%, which could meet the lowest maintenance needs to survive in winter. After analysing the nutrition constituent of the partial plants eating by feral sika deer, the crude protein content was4.7%～13.6%, the feral sika deer can self-adjust its food composition based on the practical condition. About26.4%～42.1%of the dry matter content in the feces of sika deer was different depend on the different feed and digestion ability. The fecal nitrogen content account the dry matter for1.38%～2.17%, which could not be used as the referent of food nutrition value independently. In the urine of sika deer, the ratio of urea nitrogen and the creatinine reflects the different animals' nutrition condition in different areas individually, the results revealed that the nutrition condition of the infant deer and the male mature ones were worse than the female mature ones in winter. But the negative balance and the body weight reduction in winter nutrition metabolism was a kind of normal physiological phenomenon to adapt the environment, when the ratio of urea nitrogen and the creatinine were used to determine the animal nutrition condition into undernourishment, we should be more careful, because the critical value of the normal nutrition condition of sika deer and the early-phase lacking the value of urea nitrogen and the creatinine may be6-10.
The purine derivative content in the urine of sika deer was detected by high performance liquid chromatography, the result showed that the ratio of allantoin compared with creatinine and purine derivatives compared with the creatinine in the urine of fawn is obviously higher than the mature sika deer, the proportion of Allantoin accounting for the total purine derivatives in urine was74%-89%, uric acid was2%-10%, the xanthine and hypoxanthic was8%～16%, the ratio of allantoin compared with creatinine and purine derivatives compared with the creatinine presents significant correlation.
After inspecting the parasite from110captive and feral sika deer fecal samples randomly, there was only one captive sample checked out the ball insects egg, and the quantity was quiet small, therefore we considered that sika deer has the strong anti-parasite ability.
Through the synthetical assessment, we figured that the nutrition condition of the feral sika deer living in Baiquan was pretty well, its stress and the immunity level were both in the normal range, most individuals in the feral population were healthy. The latitude of Baiquan area is high, the winter is cold, which is in the mosaic of farmland and forest, the living environment is disgusting, and the artificial activity is quite rampant, fortunately, a strong adaptiveness were showed by the sika deer in our study.
Most of the wild deer are protectional aimal in China, non-invasive method such as the fecal and uric physiology, the biochemistry target and unifies the parasite inspection etc. were useful and safety to assess its state of health in cold winter.
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