The aim of this study was to compare measurements of spermatozoal membrane status in dogs using computer-assisted spermatozoal quantification (CASQ) after staining with SYBR-14 and propidium iodide (PI) with manual counting after CFDA/PI staining. CASQ was performed on fresh (n = 11) and thawed cryopreserved canine semen (n = 91) using (1) a red long-pass (LP) filter on an untreated sample (membrane-disrupted spermatozoa, MDS count) and in a sample with all cellular membranes disrupted (total spermatozoal count, TC), (2) green LP filter for a TC and the red filter for an MDS count and (3) a green short-pass filter to obtain a membrane-intact spermatozoa (MIS) count and the red filter to obtain the MDS count, which were added to give a TC (red–green filter CASQ, n = 50). Spermatozoa were also stained with CFDA/PI, manually examined and classified as MIS or MDS. All measurements were performed in duplicate. The percentage of membrane-intact spermatozoa (MIS) was calculated. The percentage of progressively motile spermatozoa (PMS) was determined subjectively. The data were analysed to measure the agreement between the CASQ and CFDA/PI methods, repeatability of the methods and correlation between the MIS and PMS percentage. Compared with the CFDA/PI method, the agreement of MIS percentage with red filter CASQ was −12% to 34%, green LP filter CASQ −42% to 47% and red–green filter CASQ −23% to 29%. The repeatability of the CFDA/PI and red–green filter CASQ methods were the highest. The MIS and PMS percentages were always correlated (p < .05). Measurement of MIS percentage using red and red–green filter CASQ appeared to be the most reliable automated methods.

中文翻译：

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与 CFDA/PI 染色后的手动计数相比，SYBR-14/PI 染色后使用荧光计算机辅助精子定量方法测量犬精子的膜完整性

本研究的目的是比较在用 SYBR-14 和碘化丙啶 (PI) 染色后使用计算机辅助精子定量 (CASQ) 与 CFDA/PI 染色后手动计数对狗的精子膜状态的测量。CASQ 是在新鲜 ( n  = 11) 和解冻的冷冻保存的犬精液 ( n  = 91) 上进行的，使用 (1) 红色长通 (LP) 过滤器对未经处理的样本（膜破裂的精子，MDS 计数）和样本进行破坏所有细胞膜（总精子数，TC），（2）TC 的绿色 LP 过滤器和 MDS 计数的红色过滤器和（3）绿色短通过滤器，以获得膜完整的精子 (MIS)计数和红色过滤器以获得 MDS 计数，将其添加以给出 TC（红绿过滤器 CASQ，n = 50）。精子也用 CFDA/PI 染色，人工检查并分类为 MIS 或 MDS。所有测量均重复进行。计算膜完整精子（MIS）的百分比。进行性活动精子 (PMS) 的百分比是主观确定的。分析数据以衡量 CASQ 和 CFDA/PI 方法之间的一致性、方法的可重复性以及 MIS 和 PMS 百分比之间的相关性。与 CFDA/PI 方法相比，MIS 百分比与红色滤光片 CASQ 的一致性为 -12% 至 34%，绿色 LP 滤光片 CASQ 的一致性为 -42% 至 47%，红绿滤光片 CASQ 的一致性为 -23% 至 29%。CFDA/PI 和红绿滤光片 CASQ 方法的重复性最高。MIS 和 PMS 百分比始终相关（p < .05）。使用红色和红绿滤光片 CASQ 测量 MIS 百分比似乎是最可靠的自动化方法。