Freezing cooled-transported semen allows veterinarians and breeders to collect and process the semen of stallions on farm, and then ship the semen to a semen freezing center. There, however, is a lack of standardization of shipping and freezing protocols. The objectives were to optimize and simplify protocols to freeze cooled-shipped semen. In Experiment 1, cooled-transported semen was centrifuged at room temperature or 5 °C before freezing. Sperm variables (motility, membrane integrity, acrosome integrity, membrane fluidity) were evaluated before and after freezing. Centrifugation temperature had no effect on post-thaw semen quality. In Experiment 2, cooled-transported semen was centrifuged at room temperature and cryopreserved in three semen freezing extenders. With use of the improved modified French formula, there was less post-thaw total and progressive motility compared with use of Botucrio or the improved lactose-EDTA formula (P<0.0001). Semen cryopreserved in the improved modified French formula also had a lesser percentage of sperm with intact membranes compared with lactose-EDTA, and a greater percentage of sperm with capacitation-like changes compared with Botucrio (P<0.0001). In Experiment 3, semen diluted in each extender was frozen conventionally or placed directly in a -80 °C ultra-freezer. Freezing in the ultra-freezer resulted in a lesser post-thaw sperm motility, but not membrane and acrosome integrity and capacitation-like changes. In conclusion, centrifugation and addition of freezing extender to cooled transported semen can be performed at room temperature or 5 °C. The Botucrio and lactose-EDTA formula are recommended for conventional cryopreservation of cooled-transported stallion semen as compared with the modified French formula.

冷冻冷却运输的精液可以让兽医和饲养员在农场收集和处理种马的精液，然后将精液运送到精液冷冻中心。然而，缺乏运输和冷冻协议的标准化。目标是优化和简化冷冻冷冻运输精液的方案。在实验 1 中，冷却运输的精液在冷冻前在室温或 5°C 下离心。在冷冻前后评估精子变量（运动性、膜完整性、顶体完整性、膜流动性）。离心温度对解冻后精液质量没有影响。在实验 2 中，冷却运输的精液在室温下离心，并在三个精液冷冻补充剂中冷冻保存。使用改良改良法式配方，P < 0.0001)。与乳糖-EDTA 相比，在改良的改良法式配方中冷冻保存的精液中具有完整膜的精子百分比较低，与 Botucrio 相比，具有类似获能变化的精子百分比较高（P <0.0001）。在实验 3 中，在每种稀释剂中稀释的精液按常规冷冻或直接放入 -80 °C 的超低温冰箱中。在超冷冻机中冷冻会导致解冻后精子活力降低，但不会导致膜和顶体完整性以及类似获能的变化。总之，可以在室温或 5°C 下对冷却的运输精液进行离心和添加冷冻稀释剂。与改良的法国配方相比，推荐使用 Botucrio 和乳糖-EDTA 配方对冷却运输的种马精液进行常规冷冻保存。