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灵长类胚胎干细胞(ES 细胞)不仅能为研究生殖发育生物学基础理论提供良好的 模型,而且可为细胞替代治疗提供大量的供体细胞,因此具有重要的研究价值。当前 灵长类ES 细胞研究还有很多问题需要解决,如分离建立更多的胚胎干细胞系,优化培 养体系,提高ES 细胞定向分化为特定细胞的比例,研究ES 细胞自我更新和分化的机 制等。本文一方面概括了灵长类ES 细胞的研究进展,另一方面并对制备抗体,免疫外 科手术法分离灵长类胚胎内细胞团,建立猕猴ES 细胞的无饲养层、无血清培养体系和 诱导猕猴ES 细胞分化成高纯度的O2A 神经胶质前体细胞进行了研究。主要结论如下: 1)分别以猕猴脾脏淋巴细胞和人外周血单个核细胞作为免疫原,免疫日本大耳白兔, 得到免疫血清。在补体介导的细胞毒作用下,兔抗人和兔抗猕猴免疫血清可以裂解人 和猕猴囊胚滋养层细胞,从而分离出内细胞团,用于分离培养人和猕猴胚胎干细胞。2) 猕猴ES 细胞在以层粘连蛋白(laminin)为胞外基质,含转化生长因子beta1(TGFβ1) 的无血清培养基(SFM)中可以稳定的增殖至少22 代,保持不分化,并具有分化成三 个胚层细胞的能力。进一步的研究发现去除TGFβ1 后,猕猴ES 细胞出现分化,整合 素表达降低,推测TGFβ1 可能通过促进猕猴ES 细胞整合素的表达,加强其与胞外基 质的相互作用,从而维持ES 细胞的自我更新。然而猕猴ES 细胞不能在纤粘连蛋白 (fibronectin)和明胶上生长。3)无饲养层、无血清培养体系中长期培养的猕猴ES 细 胞,分化出拟胚体,14 天的拟胚体在血清中分化培养一周后,在含碱性成纤维生长因 子bFGF、表皮生长因子EGF 和胰岛素+转铁蛋白+亚硒酸钠ITS 的培养基中培养, 获得97%的O2A 胶质前体细胞,得到的O2A 细胞能够稳定增殖,并且可以自发分化 为II 型星型胶质细胞和少突胶质细胞。本实验的结果有助于猕猴ES 细胞分离建系和培 养系统的优化、推动猕猴ES 细胞自我更新和诱导为神经胶质细胞机制的研究,便于建 立ES 细胞替代治疗的猕猴模型,从而为人类ES 细胞的临床疾病治疗提供参考。

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灵长类胚胎干细胞(Es)的研究不仅对理解生殖发育生物学的基础理论、而且对实现细胞替代治疗具有重要意义。当前灵长类ES细胞还有很多问题需要解决,如ES细胞培养体系的改进、ES细胞定向分化成高纯度特定组织细胞的可能性、有效性以及分化机制等问题。本文一方面概述了灵长类ES细胞相关领域的研究进展;另一方面,以称猴胚胎干细胞(rEs)为材料,在改善rES细胞的培养体系、提高其定向分化成神经前体细胞(NPs)和少突细胞的比率进行了研究。实验采用同源称猴细胞系作为饲养层培养rES细胞,并在此基础上利用肝生长因子(HGF)和GS诱导rES细胞定向分化成高纯度的NPs和少突细胞,并评价了NPs是否具有移植功能。主要结论如下:1)四种称猴细胞系(MOF、MESF、MFG和CMESF)可作为饲养层支持rES细胞的生长,保持ES自我更新的能力和分化的多能性。而且这几种称猴饲养层支持ES细胞生长的能力存在明显的差异,进一步的研究表明这些差异主要是由于基因表达种类以及表达量上的差异而导致的。2)采用HGF和GS作为诱导因子添加到NDCM液中,诱导rES细胞分化成可移植的NPs。实验结果如下:单独的HGF或GS仅能诱导ES细胞分化成65±8.3%和69±14%NPs,而HGF和GS的联合使用NPs的比率达到88.3±8.1%,进一步纯化后获得98±1.2%的NPs。获得的NPs能在体内、外分化成三个谱系神经细胞,并能整合到大鼠脑部,发生迁移和分化。25%的NPs细胞8周后仍保持存活状态,其中65-80%的细胞发生了不同程度的迁移,而且细胞在脑部的分化种类以及数目与细胞在体内所处的微环境具有重要的关系。亚克隆实验也进一步证明采用HGF+GS获得的部分单个NPs具有神经干细胞的特性,也能在体内、外分化成三个谱系的神经细胞,而另外的却只能分化成其中的一、两种谱系的细胞。3)利用五步法,ES细胞能分化成75±6.8%的少突祖细胞和81土8.6%的成熟少突细胞。HGF通过抑制NPs的凋亡和缩短NPs的复制周期,共同促进NPs的增殖。HGF也能诱导HGF+G5获得的NPs分化成少突祖细胞和成熟少突细胞,并且促进少突细胞成熟。而且HGF促进NPs分化成少突细胞的能力受到GS的调控,单独HGF作用将导致ES细胞分化成神经元。本实验的结果为促进rES细胞相关领域的研究,以及ES细胞在神经系统疾病临床应用上提供信息,也为研究NPs和少突细胞的发育提供典型的细胞模型。

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精液的冷冻保存对家畜繁殖、人类辅助生殖以及濒危物种保护有着重要意义。然而目前精子冷冻的研究很大程度上是经验性的,并且要使用含卵黄、脱脂乳等复杂成分的稀释液。这些复杂成分不仅是潜在的污染源,也使精子冷冻中的质量控制及分析特定物质的功能变得困难。即便如此也仅有约50%的精子能够存活下来,而且存活精子的受精能力也有所下降。称猴是生物医学研究中使用广泛的灵长类动物,开展赫猴精子的冷冻保存有利于该物种的繁殖并促进基础胚胎学的研究,成功的称猴精子冷冻方法还可以为其它珍稀濒危灵长类动物所借鉴。但是目前成功的称猴精子冷冻的报道仍然很少,且精子质量检测方法并不完善。因此本文致力于建立简单有效的称猴精子冷冻方法和活率检测方法。主要结果如下:1)建立了一种用单一紫外激光同时激发Hoechst33342和碘化丙锭结合流式细胞术检测称猴精子活率的有效方法,并将此方法用于称猴精子冷冻研究。2)发现甘油的一步加入或去除与分步加入或去除对称猴精子冷冻效果没有明显影响,在此基础上发现各种糖类稀释液之间的作用差异很小,但不含糖的化学成分确定的TT稀释液对精子冷冻保护作用显著高于糖类稀释液。这可能是因为TT的渗透压(138mOsm/kg)较好地适合于猫猴精子的冻存,高渗毒害作用可能是导致称猴精子用TEST冷冻存活率低的重要原因。3)以TT为基础稀释液,探讨了精子冷冻中一些关键因素,以期进一步改进冷冻方法。结果表明精液一步稀释于含5%甘油的一倍浓度TT,平衡0.5h可得到最佳效果,总体上复苏精子的运动度可达到55%以上,质膜完整性能达到6D%左右。这种方法与传统的以含有卵黄的TTE为稀释液的称猴精子冷冻方法效果相当,复苏精子还能够成功地进行体外受精。另外,用液化精液1:2直接稀释于防冻液还能进一步提高冷冻效果。本研究建立的用化学成分确定稀释液冷冻称猴精子的简单方法,有利于提高称猴精子的冻存效率,并有助于分析特定成分在精子冷冻中的作用。而Hoechst33342和碘化丙锭双染方法,是一种简单有效的精子活率检测方法,并在多参数精子功能的流式检测中有很高应用价值。

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自从1949年Polge等研究者发现甘油的冷冻保护作用,开创了低温生物学的里程碑以来,精子冷冻保存已经逐渐成为治疗人类不育、家畜繁殖和生物多样性保护的重要手段。然而目前人类对多数物种的基本生殖生物学及其种质冷冻保存的低温生物学知识仍很缺乏。哺乳动物精子冷冻的研究很大程度上是经验性的,即使运用最现代的低温冷冻程序和仪器设备,也仅有约50%的精子能够存活下来。灵长类动物的精子对冷冻非常敏感,迄今为止应用称猴冷冻精子人工受精仅产生过一个后代。为了提高冷冻保存精子的功能和受精能力,本论文以称猴为动物模型,以冷冻复苏精一的运动度、精子质膜和顶体的完整率以及体外受精作为精子功能的检钡l指标,探讨影响动物精子冷冻保存的因素。通过研究1)建立了适合称猴精子冷冻保存的适宜方法以及冷冻复苏精子功能的检测方法,并首次成功地运用冷冻称猴精子体外受精;2)检测了不同甘油浓度和二甲亚飒浓度对冷冻复苏精子运动能力、精子质膜、精子顶体结构和受精能力的影响,确定了称猴精子冷冻保存的适宜甘油浓度;3)对比研究了不同类型渗透性防冻剂在精子冷冻保存中的作用以及对冷冻复苏精子功能的影响,发现对于称猴精子,除了甘油外,乙二醇也是一种优秀的渗透性防冻剂,而丙二醇和二甲亚矾对精子产生了较大的毒害作用,并不适合称猴精子的冷冻保存;4)研究了单糖、二糖和二糖在精子冷冻复苏过程中的保护作用,发现不同类型糖类的冷冻保护作用存在显著差异,单糖和二糖对精子的冷冻保护作用优于二糖,二糖在冷冻复苏过程中对于精子的顶体产生了破坏作用,不同类型的糖类配合使用可以提高对精子的冷冻保护效率。本研究的结果有利于优化灵长类动物精子冷冻保存的方法,包括调整防冻液的成分和冷冻降温程序,从而有助于提高精子冷冻保存的存活率。此外,还有利于深入研究哺乳动物精子冷冻损伤的机制和探讨防冻剂的冷冻保护机理.

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由于伦理和材料来源的限制,目前对灵长类早期神经发育缺乏深入地了解。与啮齿类动物相比,猕猴在遗传和生理上与人类更接近,因此猕猴胚胎干细胞(rESCs)研究具有重要的研究价值,不仅能为研究发育生物学基础理论提供良好的模型,而且可为细胞替代性治疗提供大量的供体细胞。本文以rESCs为主要研究对象,在rESCs定向分化为神经细胞的基础上着重研究神经谱系分化及调控胶质祖细胞迁移的机理。主要结论如下:1) rESCs来源的神经上皮干/前体细胞(NEPs)主要变为辐射状胶质细胞(RG)后再通过中间类型的祖细胞——神经元祖细胞(NPs)和胶质祖细胞(GPs)——分别分化为神经元和胶质细胞。同时,NEPs/RG细胞群具有早期神经管背-腹和前-后轴空间特性。NEPs/RG的维持受Notch和FGFR信号作用。此外,实验中还纯化和鉴定了猕猴胶质限定性前体细胞(GRPs)。结果表明,rESCs的神经谱系分化能够模拟体内发育过程,并与啮齿类动物早期神经谱系变化过程相似。2) 气体信号分子NO(由10μM—250μM SNP供体释放)促进rESCs来源的A2B5+/Nestin/PSA-NCAM胶质祖细胞迁移。进一步研究发现Netrin-DCC信号通路介导了NO启动的细胞迁移过程。同时,Ca2也参与调控胶质祖细胞的迁移。此外,细胞外基质和整合素α6亦可能与Netrin-DCC相互作用调控细胞迁移。结果显示,NO通过激活一个复杂的信号网络系统调控胶质祖细胞迁移。本实验的研究结果有助于揭示灵长类中枢神经系统发育的机理,同时也能为治疗神经系统退行性疾病提供阶段特异性的供体细胞。

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与啮齿类动物相比,猕猴(Macaca mulatta)在遗传和生理上与人类更接近, 因此猕猴胚胎干细胞具有重要的研究价值,不仅是研究发育生物学基础理论的良 好材料,而且可为细胞替代性治疗提供很好的同种异体移植模型。猕猴胚胎干细 胞常规培养于小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF)上,这极 大地限制了对猕猴胚胎干细胞的研究和应用,因此,需要建立猕猴胚胎干细胞的 无饲养层培养体系。 本文以猕猴胚胎干细胞系IVF3.2(本实验室自主分离建系,来源于猕猴体 外受精胚胎)为研究对象,研究了在以Matrigel (growth factor reduced, BD)作为 胞外基质,以MEF 条件培养基为培养基,以trypsin 消化传代的无饲养层培养体 系中,猕猴胚胎干细胞的生长及分化能力,并比较了猕猴胚胎干细胞IVF3.2 在 无饲养层和有饲养层两种不同培养条件下的细胞周期分布及冻存复苏效率。主要 结论如下:1)经过鉴定,在本研究建立的无饲养层培养体系中,猕猴胚胎干细 胞IVF3.2 能够保持胚胎干细胞的两个基本特征,即分化的多能性和自我更新的 无限增殖能力;2)两种培养体系下的IVF3.2 的细胞周期分布没有显著差异,均 具有胚胎干细胞独特的细胞周期结构之一,即大部分细胞处于S 期(>50%的细 胞);3)无饲养层培养的IVF3.2 冻存后能高效复苏,采用常规慢速冷冻法冻存 无饲养层培养的IVF3.2,复苏后的细胞存活率高达83%,明显高于常规培养的 IVF3.2 的冻存效率。

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Purpose and rationale The purpose of the exploratory research is to provide a deeper understanding of how the work environment enhances or constrains organisational creativity (creativity and innovation) within the context of the advertising sector. The argument for the proposed research is that the contemporary literature is dominated by quantitative research instruments to measure the climate and work environment across many different sectors. The most influential theory within the extant literature is the componential theory of organisational creativity and innovation and is used as an analytical guide (Amabile, 1997; Figure 8) to conduct an ethnographic study within a creative advertising agency based in Scotland. The theory suggests that creative people (skills, expertise and task motivation) are influenced by the work environment in which they operate. This includes challenging work (+), work group supports (+), supervisory encouragement (+), freedom (+), sufficient resources (+), workload pressures (+ or -), organisational encouragement (+) and organisational impediments (-) which is argued enhances (+) or constrains (-) both creativity and innovation. An interpretive research design is conducted to confirm, challenge or extend the componential theory of organisational creativity and innovation (Amabile, 1997; Figure 8) and contribute to knowledge as well as practice. Design/methodology/approach The scholarly activity conducted within the context of the creative industries and advertising sector is in its infancy and research from the alternative paradigm using qualitative methods is limited which may provide new guidelines for this industry sector. As such, an ethnographic case study research design is a suitable methodology to provide a deeper understanding of the subject area and is consistent with a constructivist ontology and an interpretive epistemology. This ontological position is conducive to the researcher’s axiology and values in that meaning is not discovered as an objective truth but socially constructed from multiple realties from social actors. As such, ethnography is the study of people in naturally occurring settings and the creative advertising agency involved in the research is an appropriate purposive sample within an industry that is renowned for its creativity and innovation. Qualitative methods such as participant observation (field notes, meetings, rituals, social events and tracking a client brief), material artefacts (documents, websites, annual reports, emails, scrapbooks and photographic evidence) and focused interviews (informal and formal conversations, six taped and transcribed interviews and use of Survey Monkey) are used to provide a written account of the agency’s work environment. The analytical process of interpreting the ethnographic text is supported by thematic analysis (selective, axial and open coding) through the use of manual analysis and NVivo9 software Findings The findings highlight a complex interaction between the people within the agency and the enhancers and constraints of the work environment in which they operate. This involves the creative work environment (Amabile, 1997; Figure 8) as well as the physical work environment (Cain, 2012; Dul and Ceylan, 2011; Dul et al. 2011) and that of social control and power (Foucault, 1977; Gahan et al. 2007; Knights and Willmott, 2007). As such, the overarching themes to emerge from the data on how the work environment enhances or constrains organisational creativity include creative people (skills, expertise and task motivation), creative process (creative work environment and physical work environment) and creative power (working hours, value of creativity, self-fulfilment and surveillance). Therefore, the findings confirm that creative people interact and are influenced by aspects of the creative work environment outlined by Amabile (1997; Figure 8). However, the results also challenge and extend the theory to include that of the physical work environment and creative power. Originality/value/implications Methodologically, there is no other interpretive research that uses an ethnographic case study approach within the context of the advertising sector to explore and provide a deeper understanding of the subject area. As such, the contribution to knowledge in the form of a new interpretive framework (Figure 16) challenges and extends the existing body of knowledge (Amabile, 1997; Figure 8). Moreover, the contribution to practice includes a flexible set of industry guidelines (Appendix 13) that may be transferrable to other organisational settings.

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The recognition of 3-D objects from sequences of their 2-D views is modeled by a family of self-organizing neural architectures, called VIEWNET, that use View Information Encoded With NETworks. VIEWNET incorporates a preprocessor that generates a compressed but 2-D invariant representation of an image, a supervised incremental learning system that classifies the preprocessed representations into 2-D view categories whose outputs arc combined into 3-D invariant object categories, and a working memory that makes a 3-D object prediction by accumulating evidence from 3-D object category nodes as multiple 2-D views are experienced. The simplest VIEWNET achieves high recognition scores without the need to explicitly code the temporal order of 2-D views in working memory. Working memories are also discussed that save memory resources by implicitly coding temporal order in terms of the relative activity of 2-D view category nodes, rather than as explicit 2-D view transitions. Variants of the VIEWNET architecture may also be used for scene understanding by using a preprocessor and classifier that can determine both What objects are in a scene and Where they are located. The present VIEWNET preprocessor includes the CORT-X 2 filter, which discounts the illuminant, regularizes and completes figural boundaries, and suppresses image noise. This boundary segmentation is rendered invariant under 2-D translation, rotation, and dilation by use of a log-polar transform. The invariant spectra undergo Gaussian coarse coding to further reduce noise and 3-D foreshortening effects, and to increase generalization. These compressed codes are input into the classifier, a supervised learning system based on the fuzzy ARTMAP algorithm. Fuzzy ARTMAP learns 2-D view categories that are invariant under 2-D image translation, rotation, and dilation as well as 3-D image transformations that do not cause a predictive error. Evidence from sequence of 2-D view categories converges at 3-D object nodes that generate a response invariant under changes of 2-D view. These 3-D object nodes input to a working memory that accumulates evidence over time to improve object recognition. ln the simplest working memory, each occurrence (nonoccurrence) of a 2-D view category increases (decreases) the corresponding node's activity in working memory. The maximally active node is used to predict the 3-D object. Recognition is studied with noisy and clean image using slow and fast learning. Slow learning at the fuzzy ARTMAP map field is adapted to learn the conditional probability of the 3-D object given the selected 2-D view category. VIEWNET is demonstrated on an MIT Lincoln Laboratory database of l28x128 2-D views of aircraft with and without additive noise. A recognition rate of up to 90% is achieved with one 2-D view and of up to 98.5% correct with three 2-D views. The properties of 2-D view and 3-D object category nodes are compared with those of cells in monkey inferotemporal cortex.

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This paper describes a self-organizing neural model for eye-hand coordination. Called the DIRECT model, it embodies a solution of the classical motor equivalence problem. Motor equivalence computations allow humans and other animals to flexibly employ an arm with more degrees of freedom than the space in which it moves to carry out spatially defined tasks under conditions that may require novel joint configurations. During a motor babbling phase, the model endogenously generates movement commands that activate the correlated visual, spatial, and motor information that are used to learn its internal coordinate transformations. After learning occurs, the model is capable of controlling reaching movements of the arm to prescribed spatial targets using many different combinations of joints. When allowed visual feedback, the model can automatically perform, without additional learning, reaches with tools of variable lengths, with clamped joints, with distortions of visual input by a prism, and with unexpected perturbations. These compensatory computations occur within a single accurate reaching movement. No corrective movements are needed. Blind reaches using internal feedback have also been simulated. The model achieves its competence by transforming visual information about target position and end effector position in 3-D space into a body-centered spatial representation of the direction in 3-D space that the end effector must move to contact the target. The spatial direction vector is adaptively transformed into a motor direction vector, which represents the joint rotations that move the end effector in the desired spatial direction from the present arm configuration. Properties of the model are compared with psychophysical data on human reaching movements, neurophysiological data on the tuning curves of neurons in the monkey motor cortex, and alternative models of movement control.

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BACKGROUND: HIV-1 clade C (HIV-C) predominates worldwide, and anti-HIV-C vaccines are urgently needed. Neutralizing antibody (nAb) responses are considered important but have proved difficult to elicit. Although some current immunogens elicit antibodies that neutralize highly neutralization-sensitive (tier 1) HIV strains, most circulating HIVs exhibiting a less sensitive (tier 2) phenotype are not neutralized. Thus, both tier 1 and 2 viruses are needed for vaccine discovery in nonhuman primate models. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a tier 1 simian-human immunodeficiency virus, SHIV-1157ipEL, by inserting an "early," recently transmitted HIV-C env into the SHIV-1157ipd3N4 backbone [1] encoding a "late" form of the same env, which had evolved in a SHIV-infected rhesus monkey (RM) with AIDS. SHIV-1157ipEL was rapidly passaged to yield SHIV-1157ipEL-p, which remained exclusively R5-tropic and had a tier 1 phenotype, in contrast to "late" SHIV-1157ipd3N4 (tier 2). After 5 weekly low-dose intrarectal exposures, SHIV-1157ipEL-p systemically infected 16 out of 17 RM with high peak viral RNA loads and depleted gut CD4+ T cells. SHIV-1157ipEL-p and SHIV-1157ipd3N4 env genes diverge mostly in V1/V2. Molecular modeling revealed a possible mechanism for the increased neutralization resistance of SHIV-1157ipd3N4 Env: V2 loops hindering access to the CD4 binding site, shown experimentally with nAb b12. Similar mutations have been linked to decreased neutralization sensitivity in HIV-C strains isolated from humans over time, indicating parallel HIV-C Env evolution in humans and RM. CONCLUSIONS/SIGNIFICANCE: SHIV-1157ipEL-p, the first tier 1 R5 clade C SHIV, and SHIV-1157ipd3N4, its tier 2 counterpart, represent biologically relevant tools for anti-HIV-C vaccine development in primates.

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Free-ranging mantled howling monkey (Alouatta palliata Gray) females experienced a regular estrus cycle averaging 16.3 days, demonstrated sexual skin changes, and participated in multiple matings before becoming pregnant. Gestation averaged 186 days. The average interval between births was 22.5 months. Sexual maturity occurred at approximately 36 and 42 months for females and males, respectively. Female age at first birth was about 3 1/2 years. Births were scattered during some years and clustered during others. The age, rank, and parity of the females affected infant survival. More female than male infants survived to one year of age. Increased population size was the result of immigration rather than births.

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Fecal samples from 155 mantled howling monkeys (Alouatta palliata palliata) examined at Centro Ecologico La Pacifica, Guanacaste Province, Costa Rica, revealed 75 (48%) had parasitic infections. A sampling of nine howling monkeys from Santa Rosa National Park. Costa Rica indicated only one infected animal (11%). Only three of 19 (16%) spider monkeys (Ateles geoffroyi) also from Santa Rosa were infected. Controrchis biliophilus, Trypanoxyuris minutus, unidentified strongylid eggs and Isospora sp. oocysts were found. Three monkeys from La Pacifica died and were examined for adult helminths. They were infected with Ascaris lumbricoides, C. biliophilus and T. minutus.

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The breech birth of an infant mantled howling monkey was observed on February 12, 1990. The mother assisted the successful delivery by pulling on the infant's tail and hindleg. No other members of the social group attended the mother or demonstrated any interest in the birth process. Copyright © 1991 Wiley‐Liss, Inc., A Wiley Company

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One problem with dental microwear analyses of museum material is that investigators can never be sure of the diets of the animals in question. An obvious solution to this problem is to work with live animals. Recent work with laboratory primates has shown that high resolution dental impressions can be obtained from live animals. The purpose of this study was to use similar methods to begin to document rates and patterns of dental microwear for primates in the wild. Thirty-three Alouatta palliata were captured during the wet season at Hacienda La Pacifica near Canas, Costa Rica. Dental impressions were taken and epoxy casts of the teeth were prepared using the methods of Teaford and Oyen (1989a). Scanning electron micrographs were taken of the left mandibular second molars at magnifications of 200x and 500x. Lower magnification images were used to calculate rates of wear, and higher magnification images were used to measure the size and shape of microwear features. Results indicate that, while basic patterns of dental microwear are similar in museum samples and samples of live, wild-trapped animals of the same species, ecological differences between collection locales may lead to significant intraspecific differences in dental microwear. More importantly, rates of microwear provide the first direct evidence of differences in molar use between monkeys and humans.