This study aimed to compare three methods of cell death assessment [trypan blue exclusion (TBE), propidium iodide viability assay (PIVA), and transmission electron microscopy] to evaluate fresh and frozen–thawed chicken primordial germ cells (PGCs). For this study, chicken PGCs were collected from ROSS 908 and Oravka breed hens, cryopreserved-thawed according to the protocol, and submitted for different cell death assessments. We observed significant differences between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (14.14 ± 1.27 versus 7.16 ± 1.02%, respectively) and frozen–thawed (44.00 ± 2.11 versus 33.33 ± 1.67%, respectively) samples of the Oravka breed. Moreover, significant differences (p < 0.05) between TBE and PIVA techniques in the detectable proportion of dead cells in fresh (9.20 ± 0.60 versus 5.37 ± 0.51%) samples of ROSS 908 breed were recorded. Differences may be due to methodological, sensitivity, and toxicity features of each technique tested, where TB stains cell cytoplasm of dead cells and PI penetrates and intercalates into DNA of dead cells. Therefore, we suggest using a more precise and sensitive PIVA for viability evaluation of PGCs. Further research is needed to apply various fluorochromes for more detailed cell viability evaluation.

中文翻译：

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冷冻保存的鸡原始生殖细胞中死细胞比率的显微镜评估

本研究旨在比较三种细胞死亡评估方法 [台盼蓝排除 (TBE)、碘化丙啶活力测定 (PIVA) 和透射电子显微镜]，以评估新鲜和冷冻解冻的鸡原始生殖细胞 (PGC)。在这项研究中，从 ROSS 908 和 Oravka 种母鸡中收集鸡 PGC，根据协议进行冷冻保存和解冻，并提交不同的细胞死亡评估。我们观察到 TBE 和 PIVA 技术在新鲜（分别为 14.14 ± 1.27 对 7.16 ± 1.02%）和冻融（分别为 44.00 ± 2.11 对 33.33 ± 1.67%）样本中可检测到的死细胞比例方面存在显着差异。品种。此外，显着差异（p< 记录了 TBE 和 PIVA 技术在 ROSS 908 品种的新鲜样品中可检测到的死细胞比例（9.20 ± 0.60 对 5.37 ± 0.51%）之间的差异。差异可能是由于每种测试技术的方法学、敏感性和毒性特征，其中 TB 会染色死细胞的细胞质，而 PI 会渗透并嵌入死细胞的 DNA 中。因此，我们建议使用更精确和灵敏的 PIVA 来评估 PGC 的活力。需要进一步研究以应用各种荧光染料进行更详细的细胞活力评估。