Pollard S, Anderson J C, Bah F, Mateus M, Sidhu M, Simmons Dbd
Aquatic Omics Laboratory, Department of Biology, Ontario Tech University, Oshawa, ON, Canada.
Front Genet. 2022 Mar 24;13:795348. doi: 10.3389/fgene.2022.795348. eCollection 2022.
There is global acknowledgment that humane methods in animal research are a priority, but few environmental effects monitoring programs use nonlethal methods for fish. The goal of the present study was to determine the impacts of sampling small volumes of blood in larger-bodied fish on survival and healing. In addition to evaluating survival following blood sampling, we evaluated the utility of dried blood spots as an alternative for sample processing and storage in the field. In our approach, we housed 80 rainbow trout () in our flow-through aquatic facility. We then anaesthetized using MS-222 and sampled 1 μl/g bw of blood via puncture of the caudal vasculature. We tested four different post-blood sampling treatments on the puncture wound: 1. application of liquid bandage; 2. a swab of betadine; 3. a swab of fish mucous; and 4. compared survival outcomes to a group where no post-treatment was performed (negative control). Overall, we observed 90% survival among all treatments, with the most effective approach being the negative control (100% survival). Based upon these results, we repeated the blood sampling with no-post treatment by housing 20 rainbow trout (not previously tested upon) in cages at a nearby creek and monitored survival for 2 weeks post sampling. The survival rate was 95% with full healing of the puncture site in all subjects. In addition to this, we tested the efficacy of dry blood spotting on proteomic, lipidomic and amino acid analysis as an alternative method for blood sample processing and storage. It was found that dried plasma spotting using parafilm in conjunction with a modified Bligh-Dyer extraction offered the best balance for good recovery of protein, lipid and amino acids relative to wet plasma and Noviplex dried plasma spot cards. In this article, we will present the detailed results of these combined studies and describe what we have determined to be the safest non-lethal blood sampling protocol.
全球都认可在动物研究中采用人道方法是一个优先事项，但很少有环境影响监测项目对鱼类使用非致死性方法。本研究的目的是确定在体型较大的鱼类中采集少量血液对其生存和愈合的影响。除了评估采血后的存活率外，我们还评估了干血斑作为现场样本处理和储存替代方法的效用。在我们的方法中，我们将80条虹鳟鱼饲养在我们的流水式水产养殖设施中。然后我们使用MS-222进行麻醉，并通过穿刺尾血管采集1 μl/g体重的血液。我们对穿刺伤口测试了四种不同的采血后处理方法：1. 涂抹液体创可贴；2. 用碘伏擦拭；3. 用鱼黏液擦拭；4. 将存活结果与未进行处理的组（阴性对照）进行比较。总体而言，我们观察到所有处理组的存活率为90%，最有效的方法是阴性对照（100%存活）。基于这些结果，我们通过将20条虹鳟鱼（之前未进行过测试）饲养在附近小溪的网箱中，在不进行处理的情况下重复采血，并在采血后监测2周的存活率。存活率为95%，所有受试鱼的穿刺部位完全愈合。除此之外，我们还测试了干血斑在蛋白质组学、脂质组学和氨基酸分析方面作为血液样本处理和储存替代方法的功效。结果发现，使用石蜡膜结合改良的布利格-戴尔提取法进行干血浆点样，相对于湿血浆和Noviplex干血浆点样卡，在蛋白质、脂质和氨基酸的良好回收率方面提供了最佳平衡。在本文中，我们将展示这些综合研究的详细结果，并描述我们确定的最安全的非致死性采血方案。
