Bertuzzi Mariana L, Fumuso Fernanda G, Giuliano Susana M, Miragaya Marcelo H, Gallelli María F, Carretero María I
Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal (INITRA), Universidad de Buenos Aires, Buenos Aires, Argentina.
Facultad de Ciencias Veterinarias, Cátedra de Teriogenología, Universidad de Buenos Aires, Buenos Aires, Argentina.
Reprod Domest Anim. 2020 Sep;55(9):1154-1162. doi: 10.1111/rda.13755. Epub 2020 Jul 18.
The objective of this study was to design a protocol to separate spermatozoa from seminal plasma of raw llama semen without prior enzymatic treatment using a single-layer centrifugation with Androcoll-E (AE). Two experiments were performed: (a) samples were divided into three aliquots (1 ml) that were deposited on the top of 4, 5 or 6 ml of AE and were centrifuged at 800g for 20 min and (b) samples were divided into two aliquots (1 ml) that were deposited on the top of 4 ml of AE and were centrifuged at 600g or 1,000g for 20 min. Columns of 5 and 6 ml of AE showed a total sperm motility (TM) significantly lower, while in the 4 ml column, this parameter was not different from the TM of samples before the AE treatment. The percentage of spermatozoa with intact and functional membranes, normal morphology and intact acrosomes, as well as the percentages of sperm with highly condensed chromatin, was conserved (p ˃ .05) in the three column heights and in the two centrifugation speeds evaluated. In conclusion, the different column heights of AE (4, 5 and 6 ml) and the different centrifugation speeds used (600, 800 and 1,000g) allow separating spermatozoa of raw llama semen without enzymatic treatment, preserving the evaluated sperm characteristics. However, of all the studied treatments, centrifugation in the 4 ml column of AE at 800g would be the method of choice to process raw llama semen samples destined for reproductive biotechnologies.
本研究的目的是设计一种方案，在不进行酶预处理的情况下，使用Androcoll-E（AE）单层离心法从原驼精液的精浆中分离精子。进行了两项实验：（a）将样本分成三个等分试样（1毫升），置于4、5或6毫升AE之上，以800g离心20分钟；（b）将样本分成两个等分试样（1毫升），置于4毫升AE之上，以600g或1000g离心20分钟。5毫升和6毫升AE柱显示总精子活力（TM）显著较低，而在4毫升柱中，该参数与AE处理前样本的TM没有差异。在评估的三个柱高和两种离心速度下，具有完整和功能正常的膜、正常形态和完整顶体的精子百分比，以及染色质高度浓缩的精子百分比均保持不变（p＞0.05）。总之，AE的不同柱高（4、5和6毫升）和使用的不同离心速度（600、800和1000g）能够在不进行酶处理的情况下分离原驼精液的精子，同时保留所评估的精子特征。然而，在所有研究的处理方法中，以800g在4毫升AE柱中离心将是处理用于生殖生物技术的原驼精液样本的首选方法。
