虎蛙(Ranarugulosa)是我国国家二级保护两栖类物种,具有重要的生态和经济价值。近年来,由于人类过度狩猎,污染和栖息地的丧失,虎蛙的野生种群急剧下降。为了保护野生动物资源,人工养殖虎蛙在我国发展迅速。由于人工养殖的规模越来越大,疾病在虎蛙中增加和传播。血液检查是评估动物健康状况的最直接且侵入性较小的技术。因此,获得虎蛙的正常血液学指标至关重要。这项研究的目的是研究形态计量学,虎蛙外周血细胞的微观结构和细胞化学模式。
在血细胞计数板上计算了虎蛙的血细胞数,在光学显微镜下通过千分尺测量细胞尺寸。通过Wright-Giemsa染色研究血细胞的形态和分类,通过各种细胞化学染色,包括高碘酸希夫(PAS),苏丹黑B(SBB),过氧化物酶(POX),碱性磷酸酶(AKP),酸性磷酸酶(ACP),氯乙酸AS-D萘酚酯酶(CAE)和α-萘酚乙酸酯酶(ANAE)染色。
除了红细胞和血小板,在虎蛙中发现了五种类型的白细胞:中性粒细胞,嗜酸性粒细胞,嗜碱性粒细胞,淋巴细胞和单核细胞。平均红细胞,白细胞和血小板计数分别为1.33±0.15百万/mm3,3.73±0.04×104/mm3和1.7±0.01×104/mm3。小淋巴细胞是最丰富的白细胞,其次是大淋巴细胞,中性粒细胞,嗜酸性粒细胞和单核细胞,嗜碱性粒细胞最少。嗜酸性粒细胞PAS呈强阳性,对SBB有利,POX,ACP,CAE,ANAE,而AKP染色弱阳性;嗜碱性粒细胞PAS强阳性,ACP,对SBB有利,CAE,ANAE弱阳性,对AKP是否定的,POX染色;中性粒细胞ACP强阳性,SBB,PAS阳性,POX,AKP为弱阳性,CAE和ANAE染色;单核细胞PAS阳性,SBB,ANAE,ACP为弱阳性,AKP,POX,CAE染色;大淋巴细胞和血小板PAS阳性,ACP,ANAE弱阳性,而SBB为阴性,POX,AKP,CAE;小淋巴细胞与大淋巴细胞相似,除了PAS和ACP染色强阳性。
虎蛙的血细胞类型和形态与其他两栖类动物大体相似,而它们的细胞化学模式具有一些显著的物种特异性。我们的研究可以丰富两栖动物外周血细胞形态学和细胞化学的知识,并为虎蛙的健康状况评估和疾病诊断提供基线数据。
Tiger frog (Rana rugulosa) is a national second-class protected amphibian species in China with an important ecological and economic value. In recent years, due to excessive human hunting, pollution and habitat loss, the wild population of tiger frog has declined sharply. To protect wildlife resources, the artificial breeding of tiger frogs has rapidly developed in China. Diseases are increasing and spreading among tiger frogs due to the increasing scale of artificial farming. The blood examination is the most straightforward and less invasive technique to evaluate the animal health condition. Thus, it is essential to obtain the normal hematological indicators of tiger frogs. The objective of this study was to investigate the morphometry, microstructure and cytochemical patterns of peripheral blood cells in tiger frogs.
The number of blood cells in tiger frogs was counted on a blood count board, and the cell sizes were measured by a micrometer under light microscope. The morphology and classification of blood cells were studied by Wright-Giemsa staining, and the cytochemical pateerns was investigated by various cytochemical staining including periodic acid-Schiff (PAS), Sudan black B (SBB), peroxidase (POX), alkaline phosphatase (AKP), acid phosphatase (ACP), chloroacetic acid AS-D naphthol esterase (CAE) and α-naphthol acetate esterase (ANAE) staining.
Besides erythrocytes and thrombocytes, five types of leukocytes were identified in tiger frogs: neutrophils, eosinophils, basophils, lymphocytes and monocytes. The mean erythrocyte, leukocyte and thrombocyte counts were 1.33 ± 0.15 million/mm3, 3.73 ± 0.04 × 104/mm3 and 1.7 ± 0.01 × 104/mm3, respectively. Small lymphocytes were the most abundant leukocytes, followed by large lymphocytes, Neutrophils, eosinophils and monocytes, basophils were the fewest. Eosinophils were strongly positive for PAS, positive for SBB, POX, ACP, CAE, ANAE, while weakly positive for AKP staining; basophils were strongly positive for PAS, ACP, positive for SBB, CAE, weakly positive for ANAE, negative for AKP, POX staining; neutrophils were strongly positive for ACP, SBB, positive for PAS, POX, weakly positive for AKP, CAE and ANAE staining; monocytes were positive for PAS, SBB, ANAE, weakly positive for ACP, AKP, POX, CAE staining; large lymphocytes and thrombocytes were positive for PAS, ACP, weakly positive for ANAE, while negative for SBB, POX, AKP, CAE; small lymphocytes were similar to large lymphocytes, except for strongly positive for PAS and ACP staining.
The blood cell types and morphology of tiger frogs were generally similar to those of other amphibians, while their cytochemical patterns had some notable species specificity.Our study could enrich the knowledge of peripheral blood cell morphology and
cytochemistry in amphibians, and provide baseline data for health condition evaluation and disease diagnosis of tiger frogs.