Rhesus monkey sperm penetration into zona‐free hamster ova: Comparison of preparation and culture conditions

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Abstract
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 × g for 5 min in BWW, Ham's F‐10 and TALP and allowed to swim‐up 60 min at 37° in 5% CO2 and air. Alternatively, semen was mixed 1:1 with TYB, refrigerated 20 h at 4°C, centrifuged at 100 × 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 < .001) with preincubation in TYB followed by resuspension in TALP (79%).