Álvarez-Rodríguez M, Álvarez M, Anel-López L, López-Urueña E, Manrique P, Borragán S, Morrell J M, de Paz P, Anel L
Department of Clinical and Experimental Medicine (IKE), Linköping University, Linköping, Sweden.
Animal Reproduction and Obstetrics, University of León, León, Spain; ITRA-ULE, INDEGSAL, University of León, León, Spain.
Theriogenology. 2016 Apr 1;85(6):1097-105. doi: 10.1016/j.theriogenology.2015.11.021. Epub 2015 Nov 30.
The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with this colloid showed a better resistance to freezing compared with the control sample not only for motility but also for viability.
制定一种涉及使用冷冻精液进行人工授精的特定物种保护方案，需要验证一种有效的精液冷冻方法。胶体离心法已被建议并广泛应用于选择用于人工繁殖的动物精子的有效工具。本研究的目的是比较不同的离心方法，即使用Androcoll-Bear和Percoll的单层离心法以及使用PureSperm 100的双层离心法（在两种不同的不连续梯度40%-80%和45%-90%下），用于选择新鲜棕熊精子样本。在冷冻前组中，所有选定样本均显示出较高的渐进性活力和存活率（除Percoll法的活力为43.0±5.3[P<0.05]外）；除PureSperm 45/90%外，所有胶体处理后的样本顶体受损数量较少。在解冻后组中，所有测试的离心胶体均显示出良好的减少顶体受损数量的能力。此外，PureSperm处理（45/90%）不仅在解冻后立即而且在孵育试验后导致凋亡样变化增加，这使我们认为该梯度可能对精子样本产生某种有害影响。另一方面，PureSperm处理（40/80%）在顶体受损数量方面产生了与Androcoll-Bear相似的质量保存能力，但与对照组相比有所不同（对照组，5.3±0.6；PureSperm 80，2.0±0.3；Androcoll，2.1±0.9[P<0.05]），且解冻后回收的活精子数量相对于对照组有所减少（对照组，21.2±3.1；PureSperm 80，13.7±2.7[P<0.05]）。总之，Androcoll-Bear因其操作简单、程序可靠、精子选择和冷冻能力强，是处理棕熊射精样本的有用工具。这种胶体使棕熊冻融精液的几个精子参数得到改善；与对照样本相比，用这种胶体选择的新鲜样本精子不仅在活力方面而且在存活率方面对冷冻具有更好的抵抗力。
