González-Abreu David, García-Martínez Soledad, Fernández-Espín Vanesa, Romar Raquel, Gadea Joaquín
Department of Physiology, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, Murcia, 30100, Spain.
Department of Physical Chemistry, International Excellence Campus for Higher Education and Research (Campus Mare Nostrum), University of Murcia, Murcia, 30100, Spain.
Theriogenology. 2017 Apr 1;92:14-23. doi: 10.1016/j.theriogenology.2017.01.016. Epub 2017 Jan 9.
This work was designed to study whether viscous media can improve the in vitro sperm functionality in pigs by using methylcellulose as a thickener. Viscosity of porcine oviductal fluid (POF) was compared with culture medium (Tyrode's) supplemented with methylcellulose (MET 0, 0.5 and 1% w/v). Spermatozoa were incubated in the different media (0, 1 and 2 h) and sperm motion parameters, lipid membrane disorder, plasma membrane integrity and reactive oxygen species (ROS) formation were assessed. Fertilization results were assessed i) preincubating spermatozoa in the viscous media followed by gamete coculture in a non-viscous medium; and ii) gamete coculture in the viscous media. Viscosity of POF from early luteal phase was higher than late follicular phase. Medium without methylcellulose presented constant viscosity with increased shear rate, while viscosity of the POF and media with methylcellulose was reduced by increased shear rates. Methylcellulose improved sperm linearity, straightness and the proportion of fast-linear spermatozoa. Moreover, methylcellulose increased the rate of viable spermatozoa with intact acrosome and low lipid disorder, reducing the ROS generation. Preincubation in viscous media increased the penetration rate and the mean number of spermatozoa bound to the zona pellucida (both with 0.5 and 1% MET) and reduced monospermy with 1% MET. On the other hand fertilization in the viscous media reduced penetration rate and increased monospermy. The efficiency of the IVF system was not improved with the use of viscous media. The results show the relevance of increasing viscosity thus making the in vitro media more comparable to physiological conditions.
本研究旨在通过使用甲基纤维素作为增稠剂，研究粘性介质是否能改善猪体外精子功能。将猪输卵管液（POF）的粘度与添加甲基纤维素（MET 0、0.5和1% w/v）的培养基（Tyrode氏液）进行比较。精子在不同培养基中孵育（0、1和2小时），并评估精子运动参数、脂质膜紊乱、质膜完整性和活性氧（ROS）形成。受精结果通过以下方式评估：i）将精子在粘性培养基中预孵育，然后在非粘性培养基中进行配子共培养；ii）在粘性培养基中进行配子共培养。黄体早期POF的粘度高于卵泡晚期。不含甲基纤维素的培养基在剪切速率增加时呈现恒定粘度，而POF和含甲基纤维素培养基的粘度则随剪切速率增加而降低。甲基纤维素改善了精子的线性度、直线度和快速直线运动精子的比例。此外，甲基纤维素提高了顶体完整且脂质紊乱低的活精子比例，减少了ROS的产生。在粘性培养基中预孵育可提高穿透率和与透明带结合的精子平均数量（0.5%和1% MET均如此），并降低1% MET时的单精受精率。另一方面，在粘性培养基中受精会降低穿透率并增加单精受精率。使用粘性介质并未提高体外受精系统的效率。结果表明增加粘度使体外培养基更接近生理条件具有重要意义。
