Purpose

This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing.

Methods

Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell–free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12 h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated.

Results

Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12 h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups.

Conclusion

The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

中文翻译：

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精细胞游离 DNA 作为内洛尔公牛体外生育力的潜在标记

目的

本研究旨在确定冷冻前精子样本的可冷冻性和体外生育能力的标记。

方法

精液是从九头内洛尔公牛身上采集的；一半精液用于精浆游离 DNA (cfDNA) 定量，另一半进行冷冻保存。使用计算机辅助精液分析评估精子运动，并使用流式细胞术评估新鲜和冷冻/解冻精液在解冻后 0、3、6 和 12 小时的质膜完整性和稳定性、顶体完整性、细胞凋亡和线粒体电位。冷冻/解冻的精子也用于体外胚胎生产。从每头公牛中提取 cfDNA，并对总 DNA 和无细胞线粒体 DNA (cfmtDNA) 拷贝数进行定量。每只动物的精液用于体外受精，并评估卵裂、囊胚形成和细胞计数。

结果

根据存在的 cfDNA 和 cfmDNA 浓度形成两组并进行比较：低 cfDNA 和高 cfDNA 以及低 cfmtDNA 和高 cfmtDNA。解冻后 12 小时内，所评估的大多数精子参数在各组之间没有差异。高 cfDNA 组的卵裂、第 6 天和第 7 天囊胚率以及细胞数量均高于低 cfDNA 组。cfmtDNA 观察到类似的结果，但细胞数量除外，各组之间的细胞数量相似。

结论

精浆中cfDNA的浓度和cfmtDNA的相对拷贝数不能预测精液的可冷冻性，但可以用于预测体外胚胎的产生。