Ortiz Isabel, Dorado Jesús, Morrell Jane, Gosálvez Jaime, Crespo Francisco, Jiménez Juan M, Hidalgo Manuel
Veterinary Reproduction Group, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, University of Cordoba, 14071 Cordoba, Spain.
Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Box 7054, SE-75007 Uppsala, Sweden.
J Anim Sci Biotechnol. 2017 Mar 7;8:23. doi: 10.1186/s40104-017-0155-7. eCollection 2017.
Sperm DNA fragmentation (sDF) has been proved to be an important parameter in order to predict in vitro the potential fertility of a semen sample. Colloid centrifugation could be a suitable technique to select those donkey sperm more resistant to DNA fragmentation after thawing. Previous studies have shown that to elucidate the latent damage of the DNA molecule, sDF should be assessed dynamically, where the rate of fragmentation between treatments indicates how resistant the DNA is to iatrogenic damage. The rate of fragmentation is calculated using the slope of a linear regression equation. However, it has not been studied if sDF dynamics fit this model. The objectives of this study were to evaluate the effect of different after-thawing centrifugation protocols on sperm DNA fragmentation and elucidate the most accurate mathematical model (linear regression, exponential or polynomial) for DNA fragmentation over time in frozen-thawed donkey semen.
After submitting post-thaw semen samples to no centrifugation (UDC), sperm washing (SW) or single layer centrifugation (SLC) protocols, sDF values after 6 h of incubation were significantly lower in SLC samples than in SW or UDC. Coefficient of determination (R) values were significantly higher for a second order polynomial model than for linear or exponential. The highest values for acceleration of fragmentation (aSDF) were obtained for SW, followed by SLC and UDC.
SLC after thawing seems to preserve longer DNA longevity in comparison to UDC and SW. Moreover, the fine-tuning of models has shown that sDF dynamics in frozen-thawed donkey semen fit a second order polynomial model, which implies that fragmentation rate is not constant and fragmentation acceleration must be taken into account to elucidate hidden damage in the DNA molecule.
精子DNA碎片化（sDF）已被证明是预测精液样本体外潜在生育能力的一个重要参数。胶体离心法可能是一种合适的技术，用于选择解冻后对DNA碎片化更具抵抗力的驴精子。先前的研究表明，为了阐明DNA分子的潜在损伤，应动态评估sDF，各处理之间的碎片化率表明DNA对医源性损伤的抵抗力。碎片化率通过线性回归方程的斜率计算。然而，尚未研究sDF动态是否符合该模型。本研究的目的是评估不同解冻后离心方案对精子DNA碎片化的影响，并阐明冻融驴精液中DNA碎片化随时间变化最准确的数学模型（线性回归、指数或多项式）。
将解冻后的精液样本分别进行不离心（UDC）、精子洗涤（SW）或单层离心（SLC）处理后，孵育6小时后的sDF值在SLC样本中显著低于SW或UDC样本。二阶多项式模型的决定系数（R）值显著高于线性或指数模型。SW处理的DNA碎片化加速（aSDF）值最高，其次是SLC和UDC。
与UDC和SW相比，解冻后进行SLC似乎能使DNA保存更长时间。此外，模型的微调表明，冻融驴精液中的sDF动态符合二阶多项式模型，这意味着碎片化率并非恒定不变，为阐明DNA分子中的潜在损伤，必须考虑碎片化加速情况。
