Saragusty Joseph, Hildebrandt Thomas B, Behr Britta, Knieriem Andreas, Kruse Jürgen, Hermes Robert
Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke Strasse 17, D-10315 Berlin, Germany.
Anim Reprod Sci. 2009 Oct;115(1-4):255-66. doi: 10.1016/j.anireprosci.2008.11.010. Epub 2008 Nov 20.
Reproduction in captive elephants is low and infant mortality is high, collectively leading to possible population extinction. Artificial insemination was developed a decade ago; however, it relies on fresh-chilled semen from just a handful of bulls with inconsistent sperm quality. Artificial insemination with frozen-thawed sperm has never been described, probably, in part, due to low semen quality after cryopreservation. The present study was designed with the aim of finding a reliable semen freezing protocol. Screening tests included freezing semen with varying concentrations of ethylene glycol, propylene glycol, trehalose, dimethyl sulfoxide and glycerol as cryoprotectants and assessing cushioned centrifugation, rapid chilling to suprazero temperatures, freezing extender osmolarity, egg yolk concentration, post-thaw dilution with cryoprotectant-free BC solution and the addition of 10% (v/v) of autologous seminal plasma. The resulting optimal freezing protocol uses cushioned centrifugation, two-step dilution with isothermal 285 m Osm/kg Berliner Cryomedium (BC) with final glycerol concentration of 7% and 16% egg yolk, and freezing in large volume by the directional freezing technique. After thawing, samples are diluted 1:1 with BC solution. Using this protocol, post-thaw evaluations results were: motility upon thawing: 57.2+/-5.4%, motility following 30 min incubation at 37 degrees C: 58.5+/-6.0% and following 3h incubation: 21.7+/-7.6%, intact acrosome: 57.1+/-5.2%, normal morphology: 52.0+/-5.8% and viability: 67.3+/-6.1%. With this protocol, good quality semen can be accumulated for future use in artificial inseminations when and where needed.
圈养大象的繁殖率低且幼象死亡率高，这可能共同导致种群灭绝。人工授精技术是在十年前开发的；然而，它依赖于少数几头公牛的新鲜冷藏精液，而精子质量并不稳定。从未有过关于使用冻融精子进行人工授精的描述，部分原因可能是冷冻保存后精液质量较低。本研究旨在寻找一种可靠的精液冷冻方案。筛选试验包括用不同浓度的乙二醇、丙二醇、海藻糖、二甲基亚砜和甘油作为冷冻保护剂冷冻精液，并评估缓冲离心、快速冷却至零上温度、冷冻稀释液渗透压、蛋黄浓度、解冻后用无冷冻保护剂的BC溶液稀释以及添加10%（v/v）的自体精浆。最终得到的最佳冷冻方案是采用缓冲离心、用等温285 mOsm/kg的柏林冷冻培养基（BC）进行两步稀释，最终甘油浓度为7%，蛋黄浓度为16%，并通过定向冷冻技术进行大容量冷冻。解冻后，样品用BC溶液按1:1稀释。使用该方案，解冻后的评估结果为：解冻时的活力：57.2±5.4%，在37℃孵育30分钟后的活力：58.5±6.0%，孵育3小时后的活力：21.7±7.6%，完整顶体：57.1±5.2%，正常形态：52.0±5.8%，活力：67.3±6.1%。采用该方案，可以积累高质量的精液，以便在需要时用于人工授精。
