Binor Z, Rawlins R G, Van der Ven H, Dmowski W P
Department of Obstetrics and Gynecology, Rush Medical College, Chicago, IL 60612.
Gamete Res. 1988 Jan;19(1):91-9. doi: 10.1002/mrd.1120190109.
A major problem in development of nonhuman primate in vitro fertilization is the selection of donor males and repeated collection of consistent sperm samples. In practice, collection of a viable semen sample is highly dependent on operator technique and the type of animal restraint. We report an updated method for semen collection from the rhesus monkey (Macaca mulatta), use of TES-Tris (TEST) Yolk Buffer (TYB) for prolonged sperm storage and improved results of hamster ovum penetration assay. Semen was obtained from adult males restrained with 2.0 mg/kg IM ketamine hydrochloride prior to direct penile stimulation (Grass SD-9, frequency 150, delay 9, duration 7, volts 12-18, repeat mode, twin pulse). Liquified semen was washed and centrifuged twice at 100 X g for 5 min in BWW, Ham's F-10 and TALP and allowed to swim-up 60 min at 37 degrees in 5% CO2 and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4 degrees C, centrifuged at 100 X g for 5 min, and the pellet resuspended in 1.0 ml of TALP or BWW prior to use. Hamster ova penetration was achieved with capacitated macaque sperm. Penetration was significantly improved (P less than .001) with preincubation in TYB followed by resuspension in TALP (79%).
非人灵长类动物体外受精技术发展中的一个主要问题是供体雄性的选择以及持续获取质量稳定的精子样本。在实际操作中，获取一份可用的精液样本很大程度上依赖于操作人员的技术以及动物的保定方式。我们报告了一种更新后的恒河猴（猕猴）精液采集方法，使用TES-三羟甲基氨基甲烷（TEST）卵黄缓冲液（TYB）延长精子保存时间，并改善了仓鼠卵穿透试验的结果。在直接刺激阴茎之前，给成年雄性恒河猴注射2.0mg/kg的盐酸氯胺酮进行保定（Grass SD-9，频率150，延迟9，持续时间7，电压12 - 18，重复模式，双脉冲），从而获取精液。将液化的精液在BWW、Ham's F-10和TALP中以100×g离心洗涤两次，每次5分钟，然后在37℃、5%二氧化碳和空气环境中进行60分钟的上游培养。或者，将精液与TYB按1:1混合，在4℃冷藏20小时，以100×g离心5分钟，使用前将沉淀重悬于1.0ml的TALP或BWW中。用获能的猕猴精子实现了对仓鼠卵的穿透。在TYB中预孵育后再重悬于TALP中，穿透率显著提高（P < 0.001）（79%）。
