Isolation of a Megatrypanum trypanosome from sika deer (Cervus nippon yesoensis) in Japan

A trypanosome was isolated from a sika deer (Cervus nippon yesoensis) in Hokkaido, Japan, during the primary culture of sika deer renal cells. This is the first report of isolation of a Megatrypanum trypanosome from Japanese Cervidae. The trypanosome, designated TSD1, was propagated and maintained in Eagle's modified essential medium containing 20% fetal bovine serum with sika deer renal cells as feeder. The TSD1 trypanosome was morphometrically similar to Trypanosoma cervi, which is commonly isolated from American and European deer. PCR analysis with primers for 18S ribosomal DNA and nucleotide sequencing showed that TSD1 is a member of genus Trypanosoma, subgenus Megatrypanum. Phylogenetically TSD1 is closely related to T. theileri, a common trypanosome of cattle, but is distinguishable from T. theileri by some morphometrical and biological features.     

日月山梅花鹿公鹿血清生理指标研究

对日月山养鹿场围栏放牧的10头梅花鹿公鹿的7项血清生理指标进行了测定,并与祁连白唇鹿、祁连马鹿、祁连梅花鹿的某些生理指标进行比较。结果表明,日月山梅花鹿血清总蛋白显著低于祁连白唇鹿、祁连马鹿及祁连梅花鹿(P<0.01);日月山梅花鹿血清无机磷显著低于祁连白唇鹿(P<0.01),其他血清生理指标无显著差异(P>0.05)。     

Optimizing the Conditions for In Vitro Maturation and Artificial Activation of Sika Deer (Cervus nippon hortulorum) Oocytes

With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle‐stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm ) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6‐dimethylaminopurine (6‐DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique.     

Surveillance of chronic wasting disease in sika deer, Cervus nippon, from Tokachi district in Hokkaido

Surveillance of chronic wasting disease (CWD) was conducted by performing Western blot analysis of tissue samples from 136 sika deer (Cervus nippon) killed by hunters in the Tokachi district of Hokkaido Island. No prion protein (PrPSc) associated with CWD was detected in any of the samples. To assess amino acid polymorphisms of the sika deer PrP gene, nucleotide sequencing of the PrP gene was performed. The only amino acid polymorphisms detected were 3 silent mutations at nucleotide positions 63, 225 and 408. These results suggest that sika deer in the Tokachi district are genetically homogeneous, and are not infected with CWD.     

Monitoring of chronic wasting disease using real-time quaking-induced conversion assay in Japan

There has been no report on Chronic wasting disease (CWD) cases in Japan to date; however, there is concern about the geographic spread of CWD. To clarify the CWD status in Japan, we conducted CWD monitoring using real-time quaking-induced conversion (RT-QuIC) assay which can detect the low level of CWD prions. A total of 690 obex samples collected from sika deer and Reeves’s muntjac in Hokkaido and Honshu was tested for CWD prions. No CWD-positive cases were found, suggesting that CWD is nonexistent in Japan. Our results also indicate that RT-QuIC assay is useful for continuous monitoring of CWD. Furthermore, nucleotide sequence analysis of the PrP gene revealed sika deer in Japan harbor CWD susceptible allele.     

茸鹿人工选育品种品系数量性状遗传参数的统计分析

对我国人工选育的梅花鹿和马鹿 6个品种品系的 2 0多个数量性状的遗传参数 ,进行了统计分析 ,结果表明 ,变异系数除鲜茸重的较高之外 ,其他很小 ;公鹿的留种率很低 ,茸重性状的选择差很大 ;遗传力和重复力均较高 ;世代间隔较短 ;鲜茸重的遗传进展和年改进量较大 ;梅花鹿种公鹿的种用年限较短 ,并明显低于马鹿的 ;梅花鹿品种品系间和马鹿亚种间杂交F1茸重性状和繁殖成活率性状的杂种优势率非常显著 (P <0 0 1 )。此项统计分析为茸鹿的优质高效育种、提高纯繁选育速度、杂种优势利用、杂交培育新品种和育种规化及模式提供了科学依据。     

基于GBS技术对梅花鹿、马鹿及其杂交后代基因组SNP特征的分析

旨在利用基因分型测序（genotyping by sequencing,GBS）技术对梅花鹿、马鹿及其杂交后代（F1、F2）基因组的SNP特征进行分析。本试验采用GBS技术对梅花鹿（63个）、马鹿（12个）及其杂交后代（F1代112个,F2代38个,未知类型个体1个）共226个个体的血液基因组DNA进行测序,并利用本实验室前期110只梅花鹿、197只马鹿和1只F1代杂交鹿的测序数据,以梅花鹿全基因组为参考序列进行比对分析。结果,226个个体共产生Clean data 322.683 Gb,平均每个样品1 427.802 Mb;将所有样本作为一个群体检测SNP变异,共检测出SNP位点23 943 582个,质控过滤后得到SNP位点31 630个。对31 630个SNPs使用最大似然（maximum likelihood,ML）法构建的分子进化树显示,梅花鹿、马鹿、F1及F2代区分明显。对梅花鹿和马鹿的SNPs进行比对分析,筛选出可用于鉴别马鹿、梅花鹿、F1、F2的物种特异SNP位点1 032个（马鹿特异SNP位点474个,梅花鹿特异SNP位点558个）,计算结果显示,F1代个体包含马鹿特异SNPs的比例主要在40%~60%之间,F2代个体含马鹿特异SNPs的比例主要在10%~30%之间,马鹿个体中不含梅花鹿的特异SNPs,梅花鹿中55.49%的个体不含马鹿特异SNPs,17.34%的个体含马鹿特异SNPs的比例低于1%,13.29%的个体含马鹿特异SNPs的比例在1%~10%之间,其余个体含马鹿特异SNPs的比例为10%~20%（其中有一个个体含马鹿特异SNPs的比例为33.3%）。该研究为花马杂交鹿后代的鉴定提供了可靠标记,并定量估计了F1和F2代个体含马鹿特异SNPs的比例,马鹿个体中不含梅花鹿的特异SNPs,这对梅花鹿、马鹿及其杂交后代（F1、F2）的鉴别具有重要意义。     

Phylogenetic analysis of Theileria sp. from sika deer, Cervus nippon, in Japan

The 18S rRNA genes of Theileria species detected in sika deer, Cervus nippon centralis in Yamaguchi and Cervus nippon yesoensis in Hokkaido, were analyzed. The percent identities of the nucleotide sequences of Theileria from Cervus nippon centralis and Cervus nippon yesoensis were more than 99%. The percent identities of the Theileria sp. from sika deer and Theileria sergenti, Theileria buffeli and Theileria cervi were 97, 96 and 95%, respectively. Phylogenetic analysis of the gene sequences also revealed that Theileria sp. detected from sika deer comprise a clade that is clearly distinct from the clade comprised of Theileria from cattle.     

冷冻稀释液中添加大豆卵磷脂对梅花鹿冻融精子质量的影响

【目的】 探究在冷冻稀释液中添加大豆卵磷脂代替10%卵黄对梅花鹿精液冷冻保存效果的影响,为梅花鹿人工授精体系的完善提供参考。【方法】 采用电刺激法采集梅花鹿精液,以精液冷冻稀释液中分别添加1%、2%、3%、4%和5%大豆卵磷脂代替10%卵黄作为试验组,添加20%卵黄作为对照组,分别进行各组精液冷冻保存。5 d后,进行精液解冻,检测解冻后各组精子的活力、质膜完整率、顶体完整率、线粒体活性、存活时间,筛选合适浓度的大豆卵磷脂。选取4~5岁健康雌性梅花鹿,肌肉注射300 IU孕马血清促性腺激素（PMSG）和0.4 mg氯前列醇钠进行同期发情处理,发情后第20 h用20%卵黄组与筛选出的大豆卵磷脂组冻精进行人工输精,输精后30 d使用B超检测仪检测妊娠情况,统计妊娠率。【结果】 与对照组相比,1%大豆卵磷脂组冻融后的精子活力、向前活动力、快速前进活力、活率、质膜完整率、顶体完整率及线粒体活性均显著提高（P<0.05）;随着稀释液中大豆卵磷脂浓度的增加,其冻融后精子活力、向前活动力、快速前进活力、活率、质膜完整率、顶体完整率以及线粒体活性呈下降趋势,精子存活时间也随浓度的增加而减少。1%大豆卵磷脂组冻融精子人工授精梅花鹿的妊娠率为61.11%,高于对照组、2%和3%大豆卵磷脂组,但差异均不显著（P>0.05）。【结论】 在梅花鹿精子冷冻稀释液中添加1%大豆卵磷脂替代10%卵黄,能有效提高梅花鹿冻融精子的质量,为进一步筛选新型梅花鹿精液冷冻稀释液提供理论基础。     

Interspecific association of sika deer in terrestrial animal communities of Liancheng National Nature Reserve,China

The prevention and control of invasive of alien species is an important work for nature reserves. This study analyzes the development trend of the alien species sika deer in Liancheng National Nature Reserve. From October 2019 to June 2020, 3523 valid photos and videos of terrestrial animals were acquired from 130 camera traps, and sika deer were recorded in 21 photos from 13 traps. The survival of the sika deer population was investigated by means of morphological identification, population structure analysis, species relative abundance indices, and species spatial association analysis. A total of 13 sika deer individuals were identified by camera trapping, including two kids and three subadults representing the reproductive capacity of the population. Spatially, sika deer is not associated with any local species and was outside the spatial association network of terrestrial animals in Liancheng National Nature Reserve, indicating that the sika deer population has not been integrated into the local community and has failed to perform its ecological function. It is worth noting that the reserve provides habitat suitable for sika deer and that the population has adequate reproductive capacity. Due to the lack of large apex predators in the reserve, the population size of ungulates such as sika deer, red deer, and Siberian roe deer may expand and lead to population outbreaks and the associated problems for the ecosystem. To restore large- and medium-sized carnivores and avoid the population outbreak of the species, the present challenges require immediate attention in Liancheng National Nature Reserve.     

浅谈选育梅花鹿和马鹿繁殖速度及其应用

依据统计遗传学繁殖速度的理论和方法 ,并依据品种品系鉴定时可繁殖母鹿数、母鹿的繁殖成活率及其净繁殖率 (R)和繁殖年代与成年鹿只的死淘率等 ,探讨了人工选育的双阳梅花鹿品种、长白山梅花鹿品系、西丰梅花鹿品种和塔里木马鹿品种从选育鉴定到 2 0 0 1年时的繁殖速度和该年年末繁育的鹿只存栏数 ,为制定品种 (系 )繁育计划、规范鹿种销售市场、修订和制定鹿品种审定标准 (试行 )等提供了科学依据。     

梅花鹿季节性营养规律研究

为了研究在我国饲养模式下梅花鹿营养摄入的季节性规律,选用8只雄性成年梅花鹿,在我国传统饲养模式下,定期测定梅花鹿饲料消耗及营养物质摄入水平、鹿体重变化及营养相关血液指标变化,分析梅花鹿营养季节性规律变化。结果表明,①梅花鹿夏季干物质的采食(DMI)、代谢能摄入(MEI)和可消化蛋白质摄入(DCPI)处于一年中最高水平,分别为78~80 g/(kg0.75.d-1)、0.8 MJ/(kg0.75.d-1)和8~10 g/(kg0.75.d-1);夏季梅花鹿体重平均增加约20 kg;血清蛋白质水平较高。②梅花鹿公鹿秋季发情期干物质的DMI、MEI和DCPI为一年中最低水平,分别为41~60 g/(kg0.75.d-1)、0.36~0.56 MJ/(kg0.75.d-1)和1.14~3.0 g/(kg0.75.d-1);梅花鹿在9~10月份体重平均降低约17.5 kg,血清蛋白质水平较低,营养处于负平衡状态。③梅花鹿冬季DMI、MEI和DCPI处于一年中低谷,分别为70~75 g/(kg0.75.d-1)0、.61~0.69 MJ/(kg0.75.d-1)和3.0~3.6 g/(kg0.75.d-1),体重为一年中最低水平,平均约100 kg。④在圈养条件下梅花鹿MEI[MJ/(kg0.75.d-1)]和增重G[g/(kg0.75.d-1)]间存在显著正相关(P=0.0026),直线回归方程为G=28.482 MEI-17.78(R2=0.5771,P=0.0026,n=13),维持代谢能需要量为0.624 MJ/(kg0.75.d-1),体重每增加1 g,需要生长代谢能35.11 kJ。     

Immunohistochemical localization of steroidogenic enzymes in corpus luteum of wild sika deer during early mating season

We analyzed the localization of steroidogenic enzymes (P450 scc, 3 beta HSD, P450 arom and P450 c17) in the corpora lutea of two Hokkaido sika deer (Cervus nippon yesoensis) during the early mating season. Two corpora lutea were found in each female and the timing of formation of the corpora lutea seemed different. P450 scc, and 3 beta HSD, positive luteal cells were found in both corpora lutea. The existence of two functional corpora lutea from the early mating season through pregnancy suggests that progesterone secreted by two or more corpora lutea is necessary for maintenance of pregnancy in sika deer.     

Molecular analyses of a potentially novel Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer (Cervus nippon yesoensis) in Japan

An Anaplasma species closely related to Anaplasma phagocytophilum detected in sika deer in Hokkaido, Japan was molecularly analyzed using 16S rRNA, citrate synthase (gltA), and heat-shock operon (groEL) gene sequences. Genome walking was performed to determine its complete gltA and groEL sequences (1233 bp and 1650 bp, respectively). Percent identities to the closest A. phagocytophilum sequences from the US and European strains were 98.6-98.8%, 76.5%, and 80.3-80.8% for 16S rRNA, gltA, and groEL genes, respectively. For deduced amino acid sequences, percent identities to the closest A. phagocytophilum sequences were 66.7% and 97.6% for gltA and groEL genes, respectively. Phylogenetic analyses revealed divergence from any known A. phagocytophilum strain. The lower identities and the divergent phylogenetic position of the Anaplasma sp. detected from sika deer in Japan with established A. phagocytophilum strains provide evidence of its potential novelty.     

东北地区三种鹿科动物潜在栖息地预测与保护空缺分析

栖息地是野生动物赖以生存的根本,掌握物种潜在栖息地的空间分布及其与环境因子的关系可以加强对物种的保护。鹿科(Cervidae)动物是东北地区森林等生态系统不可或缺的一部分,本研究选取东北地区3种鹿科动物马鹿(Cervus elaphus)、梅花鹿(Cervus nippon)、驼鹿(Alces alces)为研究对象,通过查阅文献、全球生物多样性信息网络数据库(GBIF)以及部分外业数据得到物种分布点,运用MaxEnt软件对3种鹿科动物进行潜在栖息地预测,模型AUC值如下:马鹿0.939,梅花鹿0.991以及驼鹿0.861。将模拟得到的潜在栖息地与东北地区保护区叠加进行保护空缺分析。模型模拟得出的潜在栖息地占整个东北地区面积:马鹿(25.85%)、梅花鹿(0.99%)以及驼鹿(13.83%);潜在栖息地被现有保护区覆盖率:马鹿(10.03%)、梅花鹿(21.70%)、驼鹿(8.85%),尚有大部分潜在栖息地未得到自然保护区覆盖。上述研究结果对这3种国家级保护鹿科动物的保护管理规划的制定提供了有益参考。     

成年梅花鹿维持能量需要的研究

选用4头成年梅花公鹿,采用回归分析法,设置不同的能量进食水平,应用北京农业大学KB-Ⅰ型呼吸测热室测定其各自的产热量,将所测得的梅花鹿的代谢能日采食量(MEI)与日产热量(HP)代入Lofgreen的曲线回归方程(1gHp=a+bMEI),求得梅花鹿的维持代谢能需要量(MEm)为516KJ/W~(0.75)kg;维持代谢能利用效率(Km)为0.707;每日产热量与食入代谢能水平呈强直线相关(P<0.01):HP/W~(0.75)=304.84+0.413MEI/W~(0.75)。     

黑麂血液生理生化指标的测定

首次检测了黑麂血液常规和生理生化等23项指标,并与鹿科梅花鹿做了比较。测定结果显示,黑麂的红细胞、血小板、血红蛋白、红细胞比容高于梅花鹿指标,而白细胞、红细胞体积、红细胞平均血红蛋白浓度、平均血红蛋白量、红细胞体积分布宽度指标低于梅花鹿指标。黑麂白细胞分类结果显示,白细胞和单核细胞约占90%;嗜酸性粒细胞和嗜碱性粒细胞所占百分比各占约2%。黑麂血清生化指标测定结果显示,总胆固醇和血糖两项指标高于梅花鹿的指标;甘油三酯(TG)及极低密度脂蛋白(VLDI-C)含量显著高于梅花鹿。检测结果反映了黑麂的机体生理机能,是进行疾病临床诊断和治疗的依据。     

Caprine herpesvirus-2 in association with naturally occurring malignant catarrhal fever in captive sika deer (Cervus nippon).

Three female sika deer from a single captive herd were submitted for postmortem examination over a 139-day period. The first 2 deer submitted were reported to have lost body mass for 20 days to 1 month before euthanasia. One of these deer had diarrhea, the other had a crusting dermatitis on the nasal planum and inner aspects of both pinnae. The third hind did not have any signs of disease before it was found seizuring and was immediately euthanatized. Microscopically, all 3 animals had a lymphocytic vasculitis typical of malignant catarrhal fever (MCF), with the most severe lesions in the brain. All 3 deer were polymerase chain reaction (PCR) positive for caprine herpesvirus 2 (CpHV-2) and were negative for ovine herpesvirus 2 (OHV-2). Two healthy goats that were housed adjacent to the deer were also PCR positive for CpHV-2 and PCR negative for OHV-2. The CpHV-2, PCR amplicons from the hinds, and the 2 healthy goats had an identical single base polymorphism. A male sika deer that was housed with the hinds and a fawn from 1 of the hinds remained asymptomatic and were PCR negative for CpHV-2. This represents the first report of mortality with MCF-like lesions in association with CpHV-2.     

纤维素酶曲添加剂对鹿茸产量及饲料消化率的影响

先后分3批次,对176头梅花鹿进行了增茸试验。结果,添喂纤维素酶曲组比对照组,鹿茸产量分别提高17.38%、26.78%和31.81%,平均每头增产129g。试验组采食量比对照组低7.48%,饲料消化率随纤维素酶曲添加水平的提高而提高。粗蛋白、酸性洗涤纤维(ADF)和中性洗涤纤维(NDF)的消化率在添喂纤维素酶曲70g/头组,分别提高9.48%(P<0.01.)、7.82%(P<0.05)和8.81%(P<0.01).试验组鹿茸氨基酸和无机元素含量都有增加的趋势。血清氨基酸除胱氨酸和组氨酸外,其它氨基酸试验组均有不同程度的提高,无机元素含量除锰外,其它元素试验组均高于对照组。     

Comparison of various reproductive status in Sika deer (Cervus nippon) using fecal steroid analysis

The feasibility of fecal steroid analysis for pregnancy diagnosis and sex determination were tested in sika deer (Cervus nippon). Feces were collected from captive sika deer in June (non-breeding season and late-pregnancy period) and October (breeding season), and also from the rectum of 24 female sika deer (19 pregnant and 5 non-pregnant females) shot as part of programs for population control in February and March (mid-pregnancy period). In mid- and late-pregnancy periods, fecal progesterone concentrations were significantly higher in pregnant female than in male and non-pregnant female deer. In October, fecal testosterone concentrations were higher in adult male deer, and no difference was found between young males and females. These results suggest that fecal steroid analysis would be a useful means for estimating pregnancy status and for detecting adult male among wild deer.