Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 79409, USA.
J Anim Sci. 2023 Jan 3;101. doi: 10.1093/jas/skad205.
Myosin heavy chain (MyHC) type and muscle fiber size are informative but time-consuming variables of interest for livestock growth, muscle biology, and meat science. The objective of this study was to validate a semi-automated protocol for determining MyHC type and size of muscle fibers. Muscle fibers obtained from the longissimus and semitendinosus of fed beef carcasses were embedded and frozen within 45 min of harvest. Immunohistochemistry was used to distinguish MyHC type I, IIA, and IIX proteins, dystrophin, and nuclei in transverse sections of frozen muscle samples. Stained muscle cross sections were imaged and analyzed using two workflows: 1) Nikon workflow which used Nikon Eclipse inverted microscope and NIS Elements software and 2) Cytation5 workflow consisting of Agilent BioTek Cytation5 imaging reader and Gen5 software. With the Cytation5 workflow, approximately six times more muscle fibers were evaluated compared to the Nikon workflow within both the longissimus (P < 0.01; 768 vs. 129 fibers evaluated) and semitendinosus (P < 0.01; 593 vs. 96 fibers evaluated). Combined imaging and analysis took approximately 1 h per sample with the Nikon workflow and 10 min with the Cytation5 workflow. When muscle fibers were evaluated by the objective thresholds of the Cytation5 workflow, a greater proportion of fibers were classified as glycolytic MyHC types, regardless of muscle (P < 0.01). Overall mean myofiber cross-sectional area was 14% smaller (P < 0.01; 3,248 vs. 3,780) when determined by Cytation5 workflow than when determined by Nikon workflow. Regardless, Pearson correlation of mean muscle fiber cross-sectional areas determined by Nikon and Cytation5 workflows was 0.73 (P < 0.01). In both workflows cross-sectional area of MyHC type I fibers was the smallest and area of MyHC type IIX fibers was the largest. These results validated the Cytation5 workflow as an efficient and biologically relevant tool to expedite data capture of muscle fiber characteristics while using objective thresholds for muscle fiber classification.
肌球蛋白重链（MyHC）类型和肌纤维大小是与家畜生长、肌肉生物学和肉类科学相关的重要信息，但需要耗费大量时间来检测。本研究旨在验证一种半自动化方法，用于确定肌肉纤维的 MyHC 类型和大小。从育肥牛胴体的背最长肌和半腱肌中取出肌肉纤维，在收获后 45 分钟内进行包埋和冷冻。使用免疫组织化学技术区分 I 型、IIA 型和 IIX 型 MyHC 蛋白、抗肌萎缩蛋白和细胞核，对冷冻肌肉样本的横切片进行染色。使用两种工作流程对染色的肌肉横切片进行成像和分析：1）尼康工作流程，使用尼康 Eclipse 倒置显微镜和 NIS Elements 软件；2）Cytation5 工作流程，包括安捷伦 BioTek Cytation5 成像阅读器和 Gen5 软件。使用 Cytation5 工作流程，与尼康工作流程相比，在背最长肌（P < 0.01；分别评估了 768 根和 129 根纤维）和半腱肌（P < 0.01；分别评估了 593 根和 96 根纤维）中，评估的肌纤维数量增加了约 6 倍。使用尼康工作流程对每个样本进行成像和分析大约需要 1 小时，而使用 Cytation5 工作流程则需要 10 分钟。当使用 Cytation5 工作流程的客观阈值评估肌纤维时，无论肌肉如何，更多的纤维被归类为糖酵解型 MyHC 类型（P < 0.01）。使用 Cytation5 工作流程确定的平均肌纤维横截面积比使用尼康工作流程确定的平均肌纤维横截面积小 14%（P < 0.01；3248 µm2 和 3780 µm2）。无论使用哪种工作流程，尼康和 Cytation5 工作流程确定的平均肌纤维横截面积之间的 Pearson 相关性为 0.73（P < 0.01）。在两种工作流程中，I 型肌纤维的横截面积最小，而 IIX 型肌纤维的横截面积最大。这些结果验证了 Cytation5 工作流程是一种高效且具有生物学相关性的工具，可以加快肌肉纤维特征的数据捕获，同时使用客观阈值对肌肉纤维进行分类。
