Ultrastructure of oocyte maturation,fertilization, and early embryo development in vitro in the Siberian tiger (Panthera tigris altaica)

The application of assisted reproduction techniques to wild cats has been stalled by a lack of basic knowledge of the reproductive biology in these species. In this study, the ultrastructure of Siberian tiger (Panthera tigris altaica) cumulus-oocyte-complexes (COCs), as well as in vitro produced (IVP) zygotes and embryos were investigated, to estimate the normality of the manipulated reproduction processes. Adult female tigers were subjected to a purified porcine pFSH/pLH stimulation treatment followed by oocyte aspiration. According to morphological appearance at the stereomicroscopical level, COCs were classified as mature, immature, or degenerated, and then allocated into the following groups: presumptively immature COCs, which were in vitro matured (IVM-group) before fixation; presumptively mature COCs, which were either fixed after retrieval (pre-IVF-group), following in vitro insemination (IVF-group) or following in vitro insemination and subsequent in vitro culture (IVC-group). All specimens were processed for light and transmission electron microscopy (TEM). Both the IVM- and pre-IVF-group included oocytes in meiotic stages ranging from prophase I to metaphase II, and some prophase I oocytes in the IVM-group were apparently in their growth phase. The IVF-group presented features of presumptive normal fertilization, but aberrations such as polynucleation were also noted. The IVC-group included cleavage stage embryos of which, however, many were polynucleated. In conclusion, the procedures used for stimulation, aspiration, and classification of COCs resulted in retrieval of a heterogeneous population of oocytes which, following IVF and IVC, displayed a high rate of developmental deviations.     

mtDNA indicates profound population structure in Indian tiger (Panthera tigris tigris)

We analyzed mtDNA polymorphisms (parts of control region, ND5, ND2, Cytb, 12S, together 902 bp) in 59 scat and 18 tissue samples from 13 Indian populations of the critically endangered Indian tiger (Panthera tigris tigris), along with zoo animals as reference. Northern tiger populations exhibit two unique haplotypes suggesting genetic isolation. Western populations from Sariska (extinct in 2004) and Ranthambore are genetically similar, such that Ranthambore could serve as a source for reintroduction in Sariska. Zoo populations maintain mitochondrial lineages that are rare or absent in the wild.     

西藏墨脱县孟加拉虎种群数量调查

孟加拉虎曾在西藏墨脱县各乡镇均有分布记录,但自2002年后该地区未见确认报道。2013—2018年间,我们利用红外相机技术、信息网络收集法和足迹鉴定法调查了墨脱县孟加拉虎的9个潜在分布区域。结果显示,墨脱县仅存1~3只孟加拉虎非定居个体,仅在旱季(每年10月至来年3月)游荡活动于背崩乡、墨脱镇雅鲁藏布江南岸区,以及格当乡金珠藏布南岸区。该区域内孟加拉虎的保护前景不容乐观,其原因是多方面的。本文提出如下建议以加强针对野生孟加拉虎的保护:(1)继续加强虎的基础性监测;(2)加强针对偷盗猎活动的野外巡护与执法管理;(3)加强对原始森林的保护;(4)改善原住民生计,加强宣传教育,以减少对自然资源的消耗;(5)恢复与建立景观廊道,改善栖息地连通性,以促进虎的自然扩散;(6)加强不同机构与参与方之间的合作研究和技术交流。     

圈养东北虎血液生理参数的测定

东北虎(Panthera tigris altaica)是一种淑临灭绝的食肉动物,现已被列入IUCN濒危物种红色名录(Goodrich et al.,2015)。东北虎主要分布于俄罗斯远东地区、中国东北地区和朝鲜的北部(Kitchener et al.,2017)。根据中俄边境地区的野生东北虎数量调查统计,该地区生存着成年老虎39只和幼虎22只(Matiukhina et al., 2016)。     

Fourteen new di- and tetranucleotide microsatellite loci for the critically endangered Indian tiger (Panthera tigris tigris)

We describe 11 dinucleotide and three tetranucleotide microsatellite loci for the critically endangered Indian tiger, Panthera tigris tigris. All of them were polymorphic with four to nine alleles per locus and an observed heterozygosity between 0.13 and 1.0. All primers also amplify microsatellite loci in leopard, Panthera pardus, and 12 primer pairs yielded reproducible results in domestic cat, Felis catus. These new microsatellites specifically developed for Indian tiger - in combination with those already available - comprise a reasonable number of loci to genetically analyse wild and captive populations of this illustrative species and might allow for recognition of individual tigers.     

Highly conserved D-loop-like nuclear mitochondrial sequences (Numts) in tiger (Panthera tigris)

Using oligonucleotide primers designed to match hypervariable segments I (HVS-1) ofPanthera tigris mitochondrial DNA (mtDNA), we amplified two different PCR products (500 bp and 287 bp) in the tiger (Panthera tigris), but got only one PCR product (287 bp) in the leopard (Panthera pardus). Sequence analyses indicated that the sequence of 287 bp was a D-loop-like nuclear mitochondrial sequence (Numts), indicating a nuclear transfer that occurred approximately 4.8–17 million years ago in the tiger and 4.6–16 million years ago in the leopard. Although the mtDNA D-loop sequence has a rapid rate of evolution, the 287-bp Numts are highly conserved; they are nearly identical in tiger subspecies and only 1.742% different between tiger and leopard. Thus, such sequences represent molecular ‘fossils’ that can shed light on evolution of the mitochondrial genome and may be the most appropriate outgroup for phylogenetic analysis. This is also proved by comparing the phylogenetic trees reconstructed using the D-loop sequence of snow leopard and the 287-bp Numts as outgroup.     

半散养东北虎繁殖期PAE编码行为谱的构建

2006—2007年,采用全事件记录法、目标动物取样法和行为取样法对泰山东北虎园半散养东北虎繁殖期的各种行为进行了观察。基于以"姿势-动作-环境"(posture-act-environment,PAE)为轴心,以行为生态功能为依据的PAE编码系统,对半散养东北虎的行为进行分类和系统编码并构建PAE行为谱。研究结果区分和定义了14种姿势、47种动作和98种行为型;并将所区分、命名和编码的所有行为型归类于摄食、排遗、调温、发情、交配、分娩、冲突、育幼、通讯、聚群、休息、运动和杂类等13类;由此构建了基于PAE编码系统的半散养东北虎首个繁殖期行为谱。     

Ultrastructure of the domestic tom cat (Felis domestica ) and tiger (Panthera tigris altaica ) spermatozoa

The ultrastructure of spermatozoa from the domestic tom cat and the Siberian tiger was studied. Semen was collected from anesthetized tom cats and Siberian tigers by electroejaculation. Spermatozoa were fixed and processed for examination by transmission electron microscopy. The principle differences between the spermatozoa from the two species were the head shape, mitochondrial organization in the neck area and structure of the fibrous sheath. Tom cat spermatozoa had an elongated oval-shaped head, while tiger spermatozoa had a more rounded head shape. Circularly oriented mitochondria in the neck area, near the proximal centriole, were frequently observed in tiger cells but rarely observed in tom cat cells. The semicircular ribs of tom cat spermatozoa were larger than the ribs of tiger spermatozoa. Also, the dense fibers (Numbers 3 and 8) of the corresponding microtubule doublets were fused or connected to the longitudinal columns in tiger spermatozoa but showed only occasional attachment in tom cat spermatozoa. These differences could influence results when the tom cat is used as a model for studying tiger semen.     

Carnivora: the amino-acid sequence of the adult Sumatran tiger (Panthera tigris sumatrae) hemoglobins

The complete amino-acid sequences of the hemoglobins from the adult Sumatran tiger (Panthera tigris sumatrae) have been determined on automatic liquid- and gas-phase sequenators. The globin chains were isolated by reverse phase HPLC on a column of Nucleosil-C4. N-Acetylserine was detected by FAB-mass spectroscopy as N-terminal aminoacid residue of the beta I chain. Comparing the sequences of the globin chains of the tiger with that of human Hb-A, 23 substitutions were recognized in the alpha, 29 in beta I and 28 in the beta II chain.     

Twenty-one novel tri- and tetranucleotide microsatellite loci for the Amur tiger (Panthera tigris altaica)

Amur tiger is the largest subspecies of tiger in the world and his conservation has also received much attention. In this study, we isolated and characterized twenty-one tri- and tetranucleotide microsatellite markers from this species. The number of alleles for each locus ranged from two to nine in a group of 60 individuals and the observed and expected heterozygosities were 0.333–0.917 and 0.302–0.822, respectively. The overall discrimination power and exclusion probabilities in parentage and paternity testing for these markers were 1.00, 0.9947 and 0.9999, respectively, indicating high-resolution power of microsatellite markers.     

圈养华南虎的个性评估及其性别差异

个性研究在动物生态研究和迁地保护中发挥着重要作用.华南虎(Panthera tigris amoyensis)作为最濒危的虎亚种,目前仍然缺乏对其个性特征的研究探索.本文通过个性特征主观评价法对6只圈养华南虎进行个性评估,并对比不同性别的华南虎个性特征的差异,使用新异物体测验进一步验证个性特征主观评价法的结果.最终提取...     

Cell cycle analysis and interspecies nuclear transfer of in vitro cultured skin fibroblasts of the Siberian tiger (Panthera tigris Altaica)

The present study was conducted to examine the effect of cell culture conditions, antioxidants, protease inhibitors (PI), and different levels of dimethylsulfoxide (DMSO) for the promotion of synchronization of different cell cycles of Siberian tiger skin fibroblasts. We also compared the ability of somatic cell nuclei of the Siberian tiger in pig cytoplasts and to support early development after reconstruction. Cell cycle synchronization between nuclear donor and recipient cells is considered to be one of the most crucial factors for successful cloning. Five experiments were performed each with a one-way completely randomized design involving three replicates of all treatments. Least significant difference (LSD) was used to determine variation among treatment groups. Experiment I focused in the effects of cycling, serum starved and fully confluent stages of Siberian tiger cells on different cell cycles. In Experiment II, the effects of different antioxidants like beta-Mercaptoethanol (beta-ME, 10 microM), cysteine (2 mM), and glutathione (2 mM) were examined after cells were fully confluent without serum starvation for 4 hr. In Experiment III, three PI, namely 6-dimethylaminopurine (6-DMAP, 2 mM), cycloheximide (7.5 microg/ml) and cytochalasin B (7.5 microg/ml) were used in the sane manner as in Experiment II. In Experiment IV, different levels of DMSO at 0%, 0.5%, 1.0%, and 2.5% were tested on different cell cycle stages of Siberian tiger examined by Flowcytometry (FACS). In Experiment I, 67.2% of the Siberian tiger skin fibroblasts reached the G0/G1 stage (2C DNA content) in fully confluent conditions which was more than the cycling (49.8%) and serum starved (SS) medium (65.5%; P < 0.05). Among the chemically treated group, glutathione (72.6%) and cycloheximide (71.3%) had little bit better results for the synchronization of G0 + G1 phases than serum starved and fully confluent. After nuclear transfer we did not see any significant differences on the development of tiger-porcine reconstructed embryos at cycling, SS and fully confluent. Data indicate that prolonged culture of cells in the absence of serum as well as using different chemicals for this experiment does not imply a shift in the percentage of cells that enter G0/G1 and that confluency is sufficient to induce quiescence. This finding can be beneficial in nuclear transfer programs in Siberian tiger, because there are negative effects, such as apoptosis associated with serum starvation.     

In-vitro induction of capacitation of fresh and frozen spermatozoa of the Siberian tiger (Panthera tigris)

Electroejaculates from 5 tigers were split and half of each was assayed fresh while the remainder was frozen and thawed before being assayed. Preincubation time, temperature and removal of seminal plasma were evaluated for their effect on in-vitro capacitation. Ability of spermatozoa to penetrate oocytes, as measured by the zona-free hamster egg-sperm penetration assay (SPA), was used as verification of capacitation. Results of the experiments with fresh semen indicate that: (1) preincubation time affects the fertilizability of tiger spermatozoa with 2 h appearing optimal, (2) a preincubation temperature of 37 degrees C results in significantly higher penetration rates than does a 22 degrees C treatment, and (3) tiger seminal plasma does not appear to contain decapacitation factors, as has been reported for several other species. Frozen semen experiments indicate that (1) frozen-thawed tiger spermatozoa must be removed from the environment of the semen extender before capacitation can take place, and (2) the freeze-thaw procedure results in a shortening of the required capacitation time.     

Interbirth interval and litter size of free-ranging Bengal tiger (Panthera tigris tigris) in dry tropical deciduous forests of India

We studied the interbirth interval (IBI) and litter size of the population of free-ranging Bengal tigers (Panthera tigris tigris) in dry tropical deciduous forests in Ranthambhore Tiger Reserve (RTR), Rajasthan, and Pench Tiger Reserve (PTR), Madhya Pradesh, between April 2005 and June 2011. Data on 15 breeding females in RTR and nine breeding females in PTR were collected using camera trapping, direct observation and radio-telemetry. The mean?±?standard error of IBI (months) in RTR was 33.4?±?3.7 and in PTR was 25.2?±?1.8. A significant difference was observed between the mean IBI of tigresses in RTR and those in PTR (df?=?9, P?=?0.04). The estimated mean litter size in RTR was 2.3?±?0.1 and that in PTR was 2.9?±?0.2. There was a significant difference between the litter size in RTR and that in PTR (χ ²?=?12.04, P?=?0.017, df?=?4). Since RTR and PTR are the important source populations of tigers in the Western and Central Indian landscapes, we propose that the tigers in these reserves be monitored, particularly for reproductive traits that are essential for understanding aspects of their population ecology.