这项工作由美国地质调查的生态系统 (切萨皮克湾环境和渔业) 和环境健康 (污染物生物学) 计划和西弗吉尼亚自然资源部资助。使用贸易名称仅用于识别目的, 并不意味着美国政府认可。

这里描述的方法已被 Leetown 科学中心的机构动物护理和使用委员会批准。
1. 收集鱼类
<ol><li>用最少的压力收集活鱼。使用船或背包 electrofishing, 钩和线或网。</li>
	<li>将鱼放在活水井或充气容器中, 直到取样。 注: 美国渔业学会已经出版了许多指南为鱼收集、处理和麻醉/安乐死26,27,28。处理鱼时要戴手套。</li>
</ol>2. 鱼尸检
<ol><li>
弄死一条鱼。
	<ol>
<li>在麻醉中放置鱼, 直到鳃盖运动停止, 鱼失去平衡。再过2–10分钟, 鱼就会被安乐死;然而, 这也可能因物种而异。 注: 鱼可以被安乐死与一些麻醉剂 (见材料表最常用)。安乐死的方法将取决于实验室测量, 将进行的组织收集29。</li>
	</ol>
</li>
	<li>
测量生物特征。
	<ol>
<li>把鱼秤到最近的克。</li>
		<li>测量鱼长到最近的毫米。<ol>
<li>当捏在一起时, 用嘴巴闭合到尾部末端测量总长度从嘴尖。</li>
			<li>测量从尾叉到鼻尖的叉子长度, 以及从鼻尖到身体末端 (尾部开始) 的标准长度。</li>
		</ol>
</li>
		<li>使用以下公式计算条件系数: 条件因子 = (总体重-性腺重量)/总长度3。 注意: 性腺重量从总体重中减去, 因为性腺对总体重有很大的贡献, 特别是在 prespawn 的雌性鱼身上。</li>
	</ol>
</li>
	<li>
获取血液样本。 注: 血液通常取自尾脉, 但也可从背主动脉或心脏穿刺30撤回。<ol>
<li>根据鱼的大小, 在1到5毫升的注射器上从尾静脉提取外周血样本, 22 或23克针。将针插入前侧线下方的尾部区域 (图 1A和1B)。向上角, 直到击中脊柱, 然后略有撤回。静脉在上覆脊柱腹侧。 注: 如果要进行血液涂片或血清是必需的, 没有抗凝剂使用。在大多数情况下, 血浆将被收集, 因此, 抗凝剂, 如肝素钠, EDTA 或锂被用来涂针和注射器, 也在采血管 (例如, vacutainer)。</li>
		<li>在将血液放入收集管之前, 取出针并放入锋利的处理容器中。 注: 血液可以在冰上举行, 但根据随后的分析应尽快离心30。</li>
		<li>如果核异常或差异性血液计数将被评估, 立刻放置一滴血液在重复的干净的玻璃显微镜幻灯片。后面的第二张幻灯片在45°角度到下降, 然后通过毛细管行动绘制横跨表面。允许空气干燥31。</li>
		<li>离心血液在 1,500–2,500 x g 15 分钟, 以沉积细胞。去除无菌转移吸管的血浆/血清, 整除进入低温瓶, 并贮存在摄氏-80 摄氏度。</li>
	</ol>
</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57946/57946fig1.jpg"> 图 1: 从鱼身上获取血液样本.(a) 最近的安乐死鱼放在其一侧和侧线处。(B) 针插入腹侧线 (箭头), 向上倾斜, 直到针接触骨干。然后, 它被轻微的撤回, 并开始抽吸血液。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<ol start="4"><li>
对每条鱼进行基于尸检的健康评估。 注: 许多发表说明和描述病变和异常的出版物可提供32、33、34、35。<ol>
<li>记录外部异常, 包括体表和鳍上的病变 (图 2), 眼睛和鳃 (图 3), 外部寄生虫如水蛭 (图 2D), 蛴螬或吸虫 metacercarial 囊肿 (图 2D,3B) 和鳃寄生虫 (图 3D)。文件类型, 在数据表上观察到的异常的位置和大小, 以及照相, 如果可能的话。</li>
		<li>用剪刀切开腹腔 (图 4A), 从肛门区域切开到笼盖, 然后取出肌肉皮瓣以暴露内脏。 注: 如果前肾脏将被收集免疫功能 (见步骤 5) 或收集的细菌学或病毒学样本, 外部身体表面应消毒70% 酒精和那些样本应该得到之前, 尸检执行。如果组织只是用于视觉观察, 血浆分析和病理学无菌技术是不必要的。</li>
		<li>记录内部异常 (图 4), 包括各器官的一般或焦点变色 (图 4B-4D)、凸起区域的存在 (图 4E)、囊肿、寄生虫和大小异常 (扩大, 萎缩)。</li>
	</ol>
</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57946/57946fig2.jpg"> 图 2: 在体表和鱼鳍上观察可见病变的例子.(a) 侧面体表的小的轻微侵蚀病灶 (箭头)。(B) 涉及尾体表面的大变红区域 (箭头)。(C) 在体表和鳍上凸起、黑色病变 (箭头)。(D) 鳍上的水蛭 (白箭) 和小黑点 (黑箭)。刻度条 = 3 毫米 (E) 在体表上有凸起的、multilobed 的、苍白的病变 (箭头)。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57946/57946fig3.jpg"> 图 3: 鱼鳃和眼睛可见病变的例子.(a) 眼睛透镜内的苍白区域 (箭头)。刻度条 = 5 毫米 (B) 白色囊肿 (白色箭头) 和小黑点 (黑色箭头), 由吸虫寄生虫引起的笼盖覆盖鳃 (a)。刻度条 = 1 厘米 (C) 鳃 (a) 上的一个苍白的、侵蚀的区域 (箭头)。刻度条 = 5 毫米 (D) 已除去的鳃, 显示附着在鳃花丝上的寄生虫 (箭头)。刻度条 = 2 毫米.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57946/57946fig4.jpg"> 图 4:鱼的尸检和内部异常的例子.(a) 在尸检期间, 鱼被切开 (沿白色箭头) 和一瓣肌肉 (黑箭头) 被去除暴露性腺 (A) 和脾脏, 由钳和剪刀举行。(b) 斑驳的肝脏 (a), 睾丸 (b), 肠周围脂肪脂肪 (c) 和胃 (d)。鳞片棒 = 5 毫米 (c) 肝 (a) 带暗红色区域 (箭头), 卵巢 (b) 和肠道 (c)。刻度条 = 5 毫米 (D) 肝带绿色变色区域 (箭头)。刻度条 = 1 厘米 (E) 正常 (a) 和异常 (b) 睾丸与凸起结节的例子。刻度条 = 1 厘米.<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<ol start="5"><li>
获得 hepatosomatic 指数 (HSI)。
	<ol>
<li>通过切断肝动脉和前端结缔组织来去除肝脏。轻轻地抬起, 同时修剪粘连和其他与肠道和脂肪脂肪的连接。当心不要刺穿胆囊。重肝。 注意: 有些鱼, 如鲤科鱼类, 没有分离的肝脏, 而是包裹在肠道和其他器官周围的肝组织。对于这些物种, 可能不可能获得肝脏重量。</li>
		<li>使用公式计算 hepatosomatic 指数 (HSI): 肝脏重量/总体重</li>
	</ol>
</li>
	<li>
计算 gonadosomatic 索引。
	<ol>
<li>移除性腺并称量它。</li>
		<li>使用公式计算 gonadosomatic 索引 (模拟器): 性腺重量/总体重</li>
	</ol>
</li>
</ol>3. 保存组织为显微病理学
注: 许多护包括10% 中性缓冲福尔马林和 Z 固定, 一种福尔马林为基础的定影剂与锌, 可用于保存在该领域的组织。如果可以使用原位杂交或荧光抗体染色等方法, 则首选后者。
<ol><li>小心切, 但不要拉出组织样本。保持单独的组织件 < 2 厘米大小和 < 5 毫米厚, 以适当的固定。作为一个经验法则, 使用大约10x 更多的固定体体积比组织, 以适当保存。将所有组织样本从一条鱼放在适当大小的同一个防漏容器中, 这取决于取样的鱼的大小。</li>
	<li>放置在固定容器中的任何外部异常件。另外, 还要包括一条相邻的正常组织。 注意: 诸如压缩或其他机械损坏、长时间暴露在空气或阳光下、冻结等不当处理会造成工件。</li>
	<li>从不同地区切开至少五3–4毫米厚的肝脏, 放入固定容器中。包括正常和异常区域, 如果观察到。</li>
	<li>根据大小, 放置一个完整的性腺或多个部分沿一个性腺进入固定容器。</li>
	<li>安置器官, 如果小, 或所有其他器官 (脾脏, 前和后肾, 鳃, 心脏, 肚腑和胃) 片断在固定容器。如果观察到异常组织, 则保留相邻的一条正常组织。</li>
</ol>4. 删除年龄分析的耳石
注意: 年龄可能是鱼类疾病/鱼类健康研究中的一个重要变量。虽然许多结构, 包括鳞片和刺, 已被用于年龄确定, 大多数研究比较结构已发现耳石给出最好的结果36,37。硬骨鱼类鱼有三对耳石-lapillus, 箭和 asteriscus。通常, 矢状或 lapillus 耳石是为衰老而收集的, 尽管可能因物种而异。去除和老化技术以前被描述了38。
<ol><li>穿过鳃峡, 然后向后弯曲。剥去在脑颅的下部分周围的结缔组织和肌肉组织, 以找到 prootic 水疱, 一个凸起的骨区。</li>
	<li>用骨切割器和裂纹切开耳石。肉眼可以看到它们。</li>
	<li>将耳石放置在标签小瓶或硬币信封中, 并在室温下存储, 直到通过计数圆环或增量38来分析年龄。如果放置在一个瓶子, 打开帽曾经返回实验室, 并允许彻底干燥之前, 储存。</li>
</ol><img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57946/57946fig5.jpg"> 图 5: 去除耳石.(A) 峡部被切断, 结缔组织和肌肉拉走, 以暴露脊柱和 neurospinal 地区的基础。(B) 骨头裂开, 露出耳石。(C) Lapillar 耳石被删除。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
5. 获得免疫功能测定用组织
注: 前肾脏是主要的造血器官, 淋巴细胞和巨噬细胞的来源为功能性化验, 必须去除灌装如果细胞将培养的功能化验, 如 mitogenesis, 吞噬和杀死能力巨噬细胞39,40。
<ol><li>用70% 乙醇喷洒鱼的外部表面。使用无菌剪刀, 手术刀和镊子打开腹腔, 并删除前肾脏组织, 这是一个黑暗的红色器官位于前的游泳膀胱。</li>
	<li>将前肾脏标本放在培养基中 (例如,莱博维茨的 L-15) 以保持细胞存活。融汇肾标本与无菌手持组织磨床 (如Tenbroeck 组织磨床) 成单细胞悬浮液。坚持湿冰, 直到返回实验室。</li>
</ol>6. 保存核酸分析用组织
注: 如果下游分子分析将进行, 如基因表达使用转录丰度41或定量 PCR42 (聚合酶链反应), 放置在适当的防腐剂评估组织片断 (例如, RNAlater 稳定解决方案) 尽快。
<ol><li>为 RNA 保存, 放置二到三小 (2–3毫米) 片断在适当的防腐剂在10:1 防腐剂容量的比率到组织。 注: 样品应保护免受阳光或过热, 并在湿冰运输。</li>
	<li>为脱氧核糖核酸保存, 安置二到三个小片断组织入95% 乙醇 (10:1 乙醇对组织按容量)。然后把样品放在湿冰上, 然后存放在-20 摄氏度。</li>
</ol>

作者没有什么可透露的。

尸检对鱼类健康的评估可以利用在任何鱼类物种上, 调查人员了解外部和内部结构的正常外观。使用标准化的方法可以比较站点和物种, 以及人口的季节性和时间变化。这些发现可用于确定与点和非定点污染源有关的效果, 并告知管理行动。它还可用于跟踪管理操作启动后的改进。可以修改方法, 以多种方式增加视觉外部异常的文档。评估仅基于视觉观察, 可以是非致命的, 相对低廉, 并且可以快速为大量的个人生成数据。因此, 它们对于探索性或初步评估是有用的, 可以监测随着时间的推移或与其他指标相结合的变化。如果在视觉观察过程中测量鱼的长度和重量, 也可以计算条件因子。虽然仅基于视觉观察的评估没有提供有关病因或相关危险因素的信息, 但某些皮肤异常45的长期趋势和生物特征参数46表明某些地区有所改善。与水质改善有关。
尸检的评估提供了更多的信息, 作为内部器官也检查和其他条件因素, 如 hepatosomatic 指数和 gonadosomatic 指数可以计算。Goede 和巴顿22开发了一个领域尸检方法, 包括血液参数, 生物统计学因素, 异常百分比和指数值的具体异常。该方法的改进包括了某些变量的严重性等级, 该值允许计算可在统计学上比较23的健康评估指数。该健康评估指数已用于区域站点比较23、47、48和其他生物指标, 包括美国地质中的血浆和组织病理学分析。调查的生物监测技术环境状况和趋势计划评估污染暴露在全国大型河流的潜在影响49,50,51。一种基于外在可见疾病和寄生虫、可见肝脏肿瘤和其他病理检测到的肝脏病变的鱼类疾病指数在北海、波罗的海和冰岛以外的海域被广泛开发和使用。该指数被发现是作为生态系统健康指标52的重要工具。
对鱼类进行基于尸检的评价, 存在一些关键因素。首先, 必须在死亡后立即对鱼类进行评估。器官颜色和一致性的变化会在死亡后相当迅速地发生。此外, 一些寄生虫可能会在死亡后不久离开宿主。第二, 重要的是要知道什么是正常的物种的利益。例如, 一些鱼通常有脂肪, 因此, 苍白的肝脏, 而对于大多数物种, 苍白的肝脏是不正常的。同样重要的是要认识到自然发生的季节性变化。在产卵季节, 有些鱼会有颜色变化或发展繁殖瘤。
基于尸检的评估作为鱼类健康评估方法的局限性包括不能 1) 一贯地确定特定病灶的 "病因" 和 2), 以确定肉眼看不到的效果。这些缺点可以克服与病理学, 分子或文化鉴定的病原体和寄生虫, 和基因表达。例如, "肿瘤" 或凸起的病变 (肿胀) 可能是实际的肿瘤, 或可能是寄生虫, 炎症, 水肿或增生 (正常细胞的数量增加), 由化学接触, 感染剂或其他刺激物引起。如代表结果所示, 明确的肿瘤或肿瘤诊断需要显微病理学来识别病变类型和严重性 (即良性或恶性)。视觉观察对白吸盘外 "肿瘤" 的评价高估了患病率, 特别是在参考点。许多突起的病变不是肿瘤, 而是增生性病变。目前尚不清楚这些增生性病变是否为前肿瘤。反之, 肝脏中凸起结节的观察明显低估了肝脏肿瘤的患病率。因此, 需要收集显微病理组织, 以充分解决除名的可能性。

人类活动对水生环境有许多不利影响。鱼类栖息于人类的各种水体中, 人们重新创建并经常用作饮水源, 因此是水生环境健康的重要指标。在特定栖息地生活和繁殖的野生鱼类在其一生中暴露于各种压力源, 包括病原体、寄生虫、水质差和化学污染物。数以千计的化学物质通过工业和人类废水、郊区/城市雨水和农业径流进入我们的水道。这些复杂的化学混合物可以有添加剂, 协同或拮抗作用对暴露的有机体1,2,3。此外, 其他环境压力源, 如高营养物质, 高温, 低溶解氧或波动 pH 可能会加剧化学污染物的影响4,5。环境压力源也可以直接影响传染病的结果, 增加6的传染性药物的数量, 增加机会病原体的毒力7或抑制免疫反应和疾病主机的抵抗8,9,10。由于这些原因, 对11、12、13、14等生物或有害影响的监测越来越感兴趣, 利用鱼类和其他水生生物来确定人口和生态系统处于危险之中。
不良影响监测利用生物标志物在不同层次的组织, 从生物体到亚细胞或分子, 以确定致死的影响, 可能影响种群和指示接触各种压力。生物水平的指标包括可见的异常和条件。计算了基于长度和重量的条件指数来评价鱼类种群的幸福度或适宜性。最常见的是富尔顿的条件因子 (K) = (重量/长度3) 15。另一个指标是可见异常的存在。在个别研究和监测程序中使用了多种方法来评估、记录和评估可见异常。仅以外部异常为基础的评估,即患有疾病、鳍损伤、肿瘤和骨骼异常的个体比例, 是评价社区健康16的生物完整性指数 (IBI) 的指标之一。类似的评估被称为 DELTs (畸形, 糜烂, 病变, 肿瘤) 也被用来评估鱼类群落的健康17。然而, 这些方法只评估外部视觉异常, 而不是内部病变或早期致死指标。
基于尸检的评估包括外部和内部观察, 并允许测量附加条件指数。Hepatosomatic 指数 (肝脏重量/总体重) 也被用作指标的健身或能量储备15 , 其中一个较高的指数值表明更健康的鱼。然而, 一些研究表明, 肥大或肝脏大小的增加是由于接触到由肝脏代谢的各种污染物18,19,20。在这种情况下, 一个较高的指数将表明接触某些化学类。gonadosomatic 指数 (性腺重量/总体重) 是另一种条件指标, 针对生殖健康21。在尸检评估期间所做的观察可以用来比较个体病变类型或正常个体百分比的患病率。然而, 它们也可以用于更定量的健康评估22,23。
此处描述的标准化尸检评估可用于根据要回答的问题、专门知识和其他可用资源, 以多种方式增加严重可见的评估。我们的例行方法是收集生物统计学数据 (长度, 体重, 肝脏重量, 性腺重量), 血液的血浆/血清分析, 文件外部和内部可见的异常, 保存部分器官的显微分析和收集耳石年龄分析。基于尸检的评估加上各种器官的年龄分析和病理组织学, 可以计算和比较各种情况指数, 可见异常的流行程度, 以及微观组织变化, 按性别、年龄、地点和取样期间。其他的组织收集可以为许多其他分析, 包括电子显微镜, 细菌学, 病毒学, 寄生虫学和化学浓度。这些方法也可以是更深入分析的一部分, 用于诊断鱼类杀死24或死亡率的圈养鱼类25的原因。方法收集组织的两个额外的分析, 基因表达和功能免疫分析。

<table><tbody><tr><td>Folding tables</td><td>Any</td><td></td><td></td></tr><tr><td>Folding chairs</td><td>Any</td><td></td><td></td></tr><tr><td>Dissecting boards</td><td>Any</td><td></td><td></td></tr><tr><td>Measuring board (mm)</td><td>Any</td><td></td><td></td></tr><tr><td>Battery powered scale (g) for fish weight</td><td>Any</td><td></td><td></td></tr><tr><td>Battery powered scale (mg) for organ weights</td><td>Any</td><td></td><td></td></tr><tr><td>Dissecting forceps</td><td>Any</td><td></td><td></td></tr><tr><td>Bone cutters</td><td>Any</td><td></td><td></td></tr><tr><td>Scalpel and blades</td><td>Any</td><td></td><td></td></tr><tr><td>Disposable gloves</td><td>Any</td><td></td><td></td></tr><tr><td>Buckets</td><td>Any</td><td></td><td></td></tr><tr><td>Leak-proof Nalgene bottles (250 mL)</td><td>ThermoFischer Scientific</td><td>02-924-5C</td><td></td></tr><tr><td>Vacutainer tubes with sodium heparin</td><td>ThermoFischer Scientific</td><td>02-689-6</td><td>For blood collection</td></tr><tr><td>Disposable&amp;nbsp; 3 mL syringes with 23 G needle</td><td>ThermoFischer Scientific</td><td>14-826-11</td><td></td></tr><tr><td>1 &amp;ndash; 2 mL cryovials</td><td>Any</td><td></td><td>Used for plasma and RNAlater samples</td></tr><tr><td>Invitrogen RNAlater Stabilization solution</td><td>ThermoFischer Scientific</td><td>AM7021</td><td></td></tr><tr><td>Z-Fix Formaldehyde Zinc fixative</td><td>Anatech LTD</td><td>SKU-174</td><td></td></tr><tr><td>Tricaine-S (MS-222)</td><td>Syndel USA</td><td></td><td>fish anesthetic</td></tr><tr><td>Coin Envelopes</td><td>Any</td><td></td><td>for otoliths</td></tr><tr><td>Pencils and pens</td><td>Any</td><td></td><td></td></tr><tr><td>70% alcohol</td><td>Any</td><td></td><td></td></tr><tr><td>Data sheets</td><td>Any</td><td></td><td></td></tr></tbody></table>

necropsy-based wild fish health assessment

人为影响从增加的营养素和化学污染物, 到栖所改变和气候变化, 可能对鱼人口有重要的影响。有害影响监测, 利用生物标志物从生物体到分子水平, 可以用来评估累积影响鱼类和其他有机体。鱼类健康已被全世界用作水生生态系统健康的指标。基于尸检的鱼类健康评估提供了可见异常和病变、寄生虫、条件和 organosomatic 指数的数据。这些可以通过地点, 季节和性别, 以及世俗的比较, 来记录时间的变化。严重性评分可以分配给各种观察, 以计算鱼类健康指数, 以进行更多的定量评估。尸检评估的一个缺点是, 它是基于视觉观察和条件因子, 它们不像组织和亚细胞生物标志物那样敏感致死作用。此外, 很少有可能发现与观察到的异常相关的原因或危险因素。例如, 在鳍、唇或体表的突起病变或 "肿瘤" 可能是肿瘤。然而, 它也可能是对寄生虫, 慢性炎症或正常细胞增生的反应, 对刺激性。相反, 肿瘤, 某些寄生虫, 其他感染性药物和许多组织改变是不可见的, 因此可能被低估。然而, 在尸检的评估期间, 血液 (血浆), 组织为病理学 (显微病理), 基因组学和其他分子分析, 和耳石为老化可以收集。这些下游分析, 连同地理空间分析、生境评估、水质和污染物分析都可以在综合生态系统评价中起重要作用。

大湖区的关切领域 (AOC) 是由于各种有益用途的损害而指定的地理区域。其中一个有益的使用损伤 (BUIs) 在许多 AOC 是鱼肿瘤或其他畸形。为了 delist 各种 BUIs 和最终的 AOC43, 已经花费了数以百万计的美元来修复和恢复这些地区。将鱼肿瘤除名的标准不同于国家 (见 epa). 政府/门户/35/lakeerie/ohio_AOC_delisting_guidance. pdf 和 dnr. 政府/专题/GreatLakes/文件/SheboyganRiverFinalReport2008. pdf);然而, 正如在除名文件中指出的, 有必要确定肝脏肿瘤的患病率, 在某些情况下是皮肤肿瘤。在许多情况下, 患病率与非 AOC 参考站点进行比较。
在三 AOCs (圣路易斯河、密尔沃基河和希博伊根河) 和一个非 AOC 参考点 (Kewaunee 河) 上, 对高级和密歇根州的鱼类肿瘤进行了评估, 利用基于尸检的白吸盘评估 (Catostomus commersonii), 其次是皮肤和肝脏组织的显微病理学。鱼从密尔沃基, 希博伊根和 Kewaunee 河收集了在2012年和 201344和从圣路易斯河在 2015年 (未发表的数据)。从密尔沃基、Kewaunee 和圣路易斯评估了200只白吸盘, 193 人来自希博伊根。
根据定义, 肿瘤可以是任何肿胀或凸起的区域, 虽然一般认为, 由异常细胞生长引起的肿胀是良性或恶性肿瘤。从所有地点收集的白色吸盘显示了各种外部凸起的病变, 包括小, 离散白点, 较大的白色区域, 轻微提高粘液病变和 multilobed 在体表和嘴唇上凸起的区域 (图 6)。对鱼类进行了称量和测量以获得条件因子, 并记录了外部和内部异常, 并收集了皮肤和肝脏组织的病理组织学。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57946/57946fig6.jpg"> 图 6: 在大湖的白吸盘上观察到皮肤病变.(a) 体表的一个离散白点。刻度条 = 5 毫米 (B) 后体表面轻微凸起的粘液 (箭头) 和 multilobed 病变 (A)。刻度条 = 1 厘米 (C) 体表的大 multilobed 病变。刻度条 = 1 厘米 (D) 唇部多裂的多重病灶。<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/57946/57946fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
与外部肿瘤或被上升的变色区域的鱼的百分比从15.5% 在圣路易斯 aoc 到58.0% 在密尔沃基 aoc。一般来说, 离散白点是最常见的视觉损害, 而 multilobed 唇和体表病变最常见。有可观察到的肝脏结节的鱼的数量是低的, 从1.5% 在 Kewaunee 和圣路易斯到2.5% 在密尔沃基 (表 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td colspan="4">河流和年份取样</td>
		</tr>
<tr>
<td>可见病变</td>
			<td>Kewaunee 2013</td>
			<td>圣路易斯2015</td>
			<td>希博伊根2012</td>
			<td>密尔沃基2013</td>
		</tr>
<tr>
<td>离散白点</td>
			<td>16</td>
			<td>3</td>
			<td>3。1</td>
			<td>5</td>
		</tr>
<tr>
<td>粘液</td>
			<td>20</td>
			<td>9。5</td>
			<td>9。8</td>
			<td>30。5</td>
		</tr>
<tr>
<td>Multilobed</td>
			<td>22。5</td>
			<td>3</td>
			<td>29。5</td>
			<td>40</td>
		</tr>
<tr>
<td>总凸起皮肤异常a
</td>
			<td>46</td>
			<td>15。5</td>
			<td>38。3</td>
			<td>58</td>
		</tr>
<tr>
<td>可见肝结节</td>
			<td>1。5</td>
			<td>1。5</td>
			<td>1。6</td>
			<td>2。5</td>
		</tr>
<tr>
<td colspan="5">
一有凸起病灶的鱼总数。有些鱼有多种类型的异常。</td>
		</tr>
</tbody></table>表 1: 以尸检为基础的观察大湖区收集的白吸盘和一个参考点 (Kewaunee 河), 以百分比表示。 目视检查可用于记录各种异常鱼类的百分比。然而, 为了明确诊断肿瘤的存在和类型, 组织必须显微镜下检查 (病理组织学)。经显微检查, 发现并非所有的凸起病变都是肿瘤。许多离散白点和粘液病变, 特别是在 Kewaunee, 是增生性病变而不是肿瘤 (表 2)。此外, 在 Kewaunee 和圣路易斯, 所有的皮肤肿瘤观察到良性 papillomas。在希博伊根和密尔沃基的 papillomas 和鳞状细胞癌, 恶性皮肤肿瘤, 观察 (表 2)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td></td>
			<td colspan="4">河流取样</td>
		</tr>
<tr>
<td>肿瘤类型</td>
			<td>Kewaunee 2013</td>
			<td>圣路易斯2015</td>
			<td>希博伊根2012</td>
			<td>密尔沃基2013</td>
		</tr>
<tr>
<td>状瘤</td>
			<td>21</td>
			<td>5。2</td>
			<td>30。5</td>
			<td>37。5</td>
		</tr>
<tr>
<td>鳞状细胞癌</td>
			<td>0</td>
			<td>0</td>
			<td>2。1</td>
			<td>10。5</td>
		</tr>
<tr>
<td>全身皮肤肿瘤</td>
			<td>21</td>
			<td>5。2</td>
			<td>32。6</td>
			<td>48</td>
		</tr>
<tr>
<td>胆管肿瘤a
</td>
			<td>2。5</td>
			<td>4</td>
			<td>6。2</td>
			<td>9。5</td>
		</tr>
<tr>
<td>肝细胞肿瘤b
</td>
			<td>1</td>
			<td>0</td>
			<td>2。1</td>
			<td>8</td>
		</tr>
<tr>
<td>全肝肿瘤</td>
			<td>3。5</td>
			<td>4</td>
			<td>8。3</td>
			<td>15.0c
</td>
		</tr>
<tr>
<td colspan="3">
一包括 cholangioma 和胆管癌</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="3">
b包括肝细胞腺瘤和肝细胞癌</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td colspan="3">
c有些鱼有胆管和肝脏肿瘤</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 2: 在大湖区收集的白色吸盘的肿瘤性病变, 以及一个参考点 (Kewaunee 河), 以百分比表示。 组织病理学分析还发现未通过视觉观察发现的肝脏肿瘤。虽然从 Kewaunee 和圣路易斯采集的鱼类中, 只有1.5% 有可见的肝脏结节 (表 1)、3.5% 和4.0% 分别有显微镜下鉴定的肿瘤 (表 2)。更大的区别被看见了在希博伊根 (1.6% 明显对8.3% 显微) 和密尔沃基 (2.5% 明显与15.0% 微观)。显微检查还提供了胆管肿瘤与肝细胞起源 (表 2) 和良性与恶性肿瘤的鉴别。

Watch this Scientific Journal Video about Necropsy-based Wild Fish Health Assessment at JoVE.com

Necropsy-based Wild Fish Health Assessment

野生鱼类的健康可以作为水生生态系统健康的指标。基于尸检的鱼类健康评估提供了可见病变或异常的文件, 用于计算条件指数的数据, 以及收集组织进行显微评估、基因表达和其他更深入的机会。分析。

基于尸检的野生鱼类健康评估

Anthropogenic influences from increased nutrients and chemical contaminants, to habitat alterations and climate change, can have significant effects on ...

necropsy-based-wild-fish-health-assessment

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

17.1K 次观看.  U.S. Geological Survey. 野生鱼类的健康可以作为水生生态系统健康的指标。基于尸检的鱼类健康评估提供了可见病变或异常的文件, 用于计算条件指数的数据, 以及收集组织进行显微评估、基因表达和其他更深入的机会。分析。

视频: Necropsy-based Wild Fish Health Assessment

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

对污水处理工艺的疗效评估利用化学和生态毒理学方法的电池，以消除雌激素潜能

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

科研

环境科学

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

绿松石鳉鱼的急性和慢性毒性测试协议Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

Modeling the Size Spectrum for Macroinvertebrates and Fishes in Stream Ecosystems

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological community or food web. Importantly, the size spectrum assumes that individual size, rather than species&#8217; behavioral or life history characteristics, is the primary determinant of abundance within an ecosystem. Thus, unlike traditional allometric relationships that focus on species-level data (e.g., mean species&#8217; body size vs. population density), size spectra analyses are &#8216;ataxic&#8217; &#8211; individual specimens are identified only by their size, without consideration of taxonomic identity. Size spectra models are efficient representations of traditional, complex food webs and can be used in descriptive as well as predictive contexts (e.g., predicting responses of large consumers to changes in basal resources). Empirical studies from diverse aquatic ecosystems have also reported moderate to high levels of similarity in size spectra slopes, suggesting that common processes may regulate the abundances of small and large organisms in very different settings. This is a protocol to model the community-level size spectrum in wadable streams. The protocol consists of three main steps. First, collect quantitative benthic fish and invertebrate samples that can be used to estimate local densities. Second, standardize the fish and invertebrate data by converting all individuals to ataxic units (i.e., individuals identified by size, irrespective of taxonomic identity), and summing individuals within log2 size bins. Third, use linear regression to model the relationship between ataxic M and D estimates. Detailed instructions are provided herein to complete each of these steps, including custom software to facilitate D estimation and size spectra modeling.

This is a protocol to model the size spectrum (scaling relationship between individual mass and population density) for combined fish and invertebrate data from wadable streams and rivers. Methods include: field techniques to collect quantitative fish and invertebrate samples; lab methods to standardize the field data; and statistical data analysis.

对河流生态系统中巨脊椎动物和鱼类的大小谱进行建模

The size spectrum is an inverse, allometric scaling relationship between average body mass (M) and the density (D) of individuals within an ecological ...

Behavioral Tracking and Neuromast Imaging of Mexican Cavefish

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among these traits, foraging behavior is one of the useful windows into functional advantages of behavioral trait evolution. Presented herein are updated methods for analyzing vibration attraction behavior (VAB: an adaptive foraging behavior) and imaging of associated mechanosensors of cave-adapted tetra, Astyanax mexicanus. In addition, methods are presented for high-throughput tracking of a series of additional cavefish behaviors including hyperactivity and sleep-loss. Cavefish also show asociality, repetitive behavior and higher anxiety. Therefore, cavefish serve as an animal model for evolved behaviors. These methods use free-software and custom-made scripts that can be applied to other types of behavior. These methods provide practical and cost-effective alternatives to commercially available tracking software.

Here, we present methods for high-throughput study of a series of the Mexican cavefish behaviors and vital staining of a mechanosensory system. These methods use free-software and custom-made scripts, providing a practical and cost-effective method for the studies of behaviors.

墨西哥辣鱼的行为跟踪与神经吻合

Cave-dwelling animals have evolved a series of morphological and behavioral traits to adapt to their perpetually dark and food-sparse environments. Among ...

行为学

Swimming Performance Assessment in Fishes

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.

The lives of the majority of fish are predicated on swimming. This protocol describes techniques for capturing a range of swimming modes available to individual and schooling fish, and includes metrics associated with swimming physiology and behaviour.

鱼类游泳绩效评估

Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, ...

生物学

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

成年斑马鱼的游泳耐力和游泳行为评估

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

神经科学

Isolation, Fixation and Characterization of Juvenile Gilthead Seabream Head Kidney Leukocytes by Flow Cytometry

Immunity is crucial for the physiological regulation of organisms, serving as the primary defense against pathogens and environmental stressors. The isolation and analysis of immune cells provide key insights into immune responses to external pressures. However, the lack of harmonized protocols for less studied species, such as marine fish, often leads to technical and analytical challenges that hamper data interpretation and a thorough understanding of species-specific immune responses. This study aimed to set up an optimized flow cytometry-based analytical procedure to characterize and determine the viability of leukocytes from the head kidney (the main hematopoietic organ in teleost fish) of juvenile gilthead seabream (Sparus aurata). The procedure began with the isolation of leukocytes through a homogenization process using Hanks&#39; balanced salt solution, followed by an optimized Percoll density gradient centrifugation method to ensure high recovery rates of leukocytes with minimal erythrocyte contamination required for efficient subsequent flow cytometry analysis. Additionally, a novel technique using a cell-reactive dye (LIVE/DEAD Fixable Dead Cell Stain Kit) was employed to distinguish viable from dead cells based on their fluorescent staining patterns. Fixation was achieved with 3.7% formaldehyde, preserving cell morphology, viability, and staining efficiency. Flow cytometry analysis successfully identified three predominant leukocyte populations: lymphocytes, monocytes, and granulocytes. This method not only allowed viability tests but also the accurate differentiation of cell types. The improvement in flow cytometry protocols represents a step forward in fish immunology by increasing the accuracy and efficiency of immune cell analysis. Furthermore, by allowing the fixation of cells for later analysis, this protocol significantly reduces the time and effort required for immune assessments, making it a valuable tool for both research and practical applications in various fields of research.

This manuscript describes the isolation and fixation of leukocytes extracted from gilthead seabream's head-kidney and the assessment of their viability by flow cytometry. This work contributes to the standardization of protocols and leverages the processing of a higher number of samples without compromising sample quality, promoting advancements in fish immunology knowledge.

通过流式细胞术分离、固定和表征幼年 Gilthead 海鲷头肾白细胞

Immunity is crucial for the physiological regulation of organisms, serving as the primary defense against pathogens and environmental stressors. The ...

免疫与感染

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.