Guillén Palomino Crissthel Yverlin, Fumuso Fernanda Gabriela, Bertuzzi Mariana Lucía, Giuliano Susana María, Velásquez González Nicolás, Bariani Maria Victoria, Carretero María Ignacia
Laboratorio de Biotecnología Reproductiva, Estación Experimental Agraria Canaán, Instituto Nacional de Innovación Agraria (INIA-Ayacucho), La Molina, Perú.
Cátedra de Teriogenología, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnología en Reproducción Animal, Universidad de Buenos Aires, Buenos Aires, Argentina.
Front Vet Sci. 2021 Jan 21;7:594926. doi: 10.3389/fvets.2020.594926. eCollection 2020.
It is not easy to separate frozen-thawed South American camelid sperm from seminal plasma (SP) and diluents to be used for embryo production. The objective of this study was to evaluate Androcoll-E™ (AE) efficiency to separate llama sperm from SP and freezing extender in frozen-thawed semen. A total of 22 ejaculates from five males were collected using electroejaculation. After performing semen analysis (sperm motility, concentration, viability, membrane function, and acrosome integrity), samples were cryopreserved with a diluent containing lactose, ethylenediaminetetraacetic acid (EDTA), egg yolk, and 7% dimethylformamide. After thawing, samples were divided in aliquots, one of which was used as a control and the others processed by AE. (12 ejaculates): 100 μl of frozen-thawed semen was placed on top of 1,000 μl AE column and centrifuged at 800 for 10 min. (10 ejaculates): two samples of 100 μl of frozen-thawed semen were placed on two columns of 500 μl AE each, and both were centrifuged at 800 for 10 and 20 min, respectively. Pellets were resuspended in Tyrode's albumin lactate pyruvate (TALP) medium, and sperm parameters were evaluated. A significant decrease in all sperm parameters was observed in thawed samples compared to raw semen. AE allowed the separation of frozen-thawed sperm from SP and freezing extender independently from the height of the column used and time of centrifugation assayed. Although no significant differences were found between AE columns, higher sperm recovery was observed with 500 μl of AE coupled with 20 min of centrifugation. Despite the significant decrease observed in sperm motility in AE samples, no changes in sperm viability, membrane function, and acrosome integrity were observed when comparing control thawed semen with the sperm recovered after AE ( > 0.05). The use of AE columns, either 500 or 1,000 μl, allows the separation of frozen-thawed llama sperm from SP and freezing extender, preserving the viability, membrane function, and acrosome integrity. Of the protocols studied, 800 centrifugation during 20 min using a 500 μl column of AE would be the method of choice to process frozen-thawed llama semen destined for reproductive biotechnologies.
将冻融后的南美骆驼科动物精子与精浆（SP）及稀释剂分离，用于胚胎生产并非易事。本研究的目的是评估Androcoll-E™（AE）从冻融精液中的精浆和冷冻稀释液中分离美洲驼精子的效率。使用电刺激采精法从5只雄性动物身上共采集了22份射精样本。在进行精液分析（精子活力、浓度、存活率、膜功能和顶体完整性）后，样本用含有乳糖、乙二胺四乙酸（EDTA）、蛋黄和7%二甲基甲酰胺的稀释液进行冷冻保存。解冻后，将样本分成若干份，其中一份用作对照，其他样本用AE处理。（12份射精样本）：将100μl冻融精液置于1000μl AE柱顶部，以800离心10分钟。（10份射精样本）：将两份100μl冻融精液样本分别置于两根500μl AE柱上，分别以800离心10分钟和20分钟。将沉淀重悬于台氏白蛋白乳酸丙酮酸（TALP）培养基中，并评估精子参数。与新鲜精液相比，解冻后的样本中所有精子参数均显著下降。AE能够从精浆和冷冻稀释液中分离冻融后的精子，且与所用柱的高度和离心时间无关。尽管AE柱之间未发现显著差异，但500μl AE结合20分钟离心可观察到更高的精子回收率。尽管AE样本中的精子活力显著下降，但将对照解冻精液与AE处理后回收的精子进行比较时，未观察到精子存活率、膜功能和顶体完整性的变化（>0.05）。使用500或1000μl的AE柱均可从精浆和冷冻稀释液中分离冻融后的美洲驼精子，同时保持其存活率、膜功能和顶体完整性。在所研究的方案中，如果要处理用于生殖生物技术的冻融美洲驼精液，选择使用500μl AE柱在800下离心20分钟的方法为宜。
