Proceedings of the 5th International Symposium on EQUINE ...

Equine Embryo TransferCRYOPRESERVATION OF EQUINE EMBRYOS BY OPS,CRYOLOOP, OR CONVENTIONAL SLOW COOLINGMETHODSN. Oberstein, M. K. O’Donovan, J. E. Bruemmer, M. Lane, G. E. Seidel, Jr.,E. M. Carnevale and E. L. SquiresAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USA; Colorado Center for Reproductive Medicine, Englewood, Colorado 80110, USACryopreservation <strong>of</strong> equine embryos has been onlymarginally successful with conventional slowcoolingprocedures. The objective <strong>of</strong> thisexperiment was to compare <strong>the</strong> efficacy <strong>of</strong> 2vitrification procedures to conventional slowcoolingprocedures for freezing equine embryos.Small (≥ 300 µm) embryos were recoveredfrom mares on Day 6 or 7 post ovulation. TwentysevenGrade 1 or 2 embryos were randomlyassigned to one <strong>of</strong> 3 cryopreservation treatments.For Treatment 1, embryos were placed in 1.8M ethylene glycol (EG) and 0.1 M sucrose andequilibrated on a warm plate at 30°C for 10 min.Individual embryos were loaded into 0.25 mlFrench straws as follows: 5 µl <strong>of</strong> H-SOF, airbubble, 50 µl H-SOF with 1.8 M EG and 0.1 Msucrose containing <strong>the</strong> embryo, air bubble, and 150µl <strong>of</strong> H-SOF. Straws were heat-sealed on one endand sealed with <strong>the</strong> PVC cotton plug on <strong>the</strong> o<strong>the</strong>rend. Straws were placed in a programmablefreezer at 0°C, cooled at 0.5°C/min to -6°C andseeded. The temperature was held for 1 min at-6°C, and <strong>the</strong>n dropped to -35°C at 0.3°C/min afterwhich straws were plunged into liquid nitrogen.Embryos were thawed by holding straws in air for10 s and <strong>the</strong>n placing <strong>the</strong>m in a 37°C waterbath for20 s. Cryoprotectant was removed by transferringembryos directly into H-SOF. Embryos weresubsequently washed through three 150 µl drops <strong>of</strong>H-SOF for 5 min each.Embryos in Treatment 2 were cryopreserved inan open pulled straw (OPS) (Hochi et al. 1996).After washing, embryos were placed into a droplet<strong>of</strong> H-SOF containing 7.5% DMSO and 7.5% EGfor 3 min. Embryos were transferred inapproximately 1–2 µl <strong>of</strong> solution into a 20 µldroplet <strong>of</strong> H-SOF with 16.5% EG, 16.5% DMSOand 0.5 M sucrose. Approximately l µl <strong>of</strong> mediumcontaining <strong>the</strong> embryo <strong>the</strong>n was drawn into <strong>the</strong>fine end <strong>of</strong> an open pulled straw by capillaryaction. The fine end was immediately submergedinto liquid nitrogen. The interval from embryocontact with concentrated cryoprotectant solutionand freezing was 20–30 s. Straws were stored inliquid nitrogen until viability testing. Embryoswere thawed by immersing <strong>the</strong> pulled end <strong>of</strong> <strong>the</strong>straw in 1.2 ml H-SOF containing 0.15 M sucrosefor 5 min and moved twice into fresh H-SOF for 5min.For Treatment 3, embryos were directlyimmersed into liquid nitrogen using a cryoloop(Hochi et al. 1994). Embryos were washed 4times in H-SOF and placed into H-SOF with 7.5%DMSO and 7.5% EG for 2.5 min. Embryos weretransferred into H-SOF with 17.5% DMSO, 17.5%EG, 1 M sucrose, and 0.25 µM ficoll. A nylon loopwas mounted. Embryos were transferred into H-SOF with 17.5% DMSO, 17.5% EG, 1 M sucrose,and 0.25 µM ficoll. on a stainless steel cylinderinserted into <strong>the</strong> lid <strong>of</strong> a cryovial. Loops werepurchased mounted and epoxied into vials. Themetal insert on <strong>the</strong> lid enabled <strong>the</strong> use <strong>of</strong> a handlewith a small magnet for manipulation <strong>of</strong> <strong>the</strong> loop.While <strong>the</strong> embryo was in <strong>the</strong> first cryoprotectantsolution, <strong>the</strong> cryoloop was dipped into <strong>the</strong> secondcryoprotectant solution to retain a thin film <strong>of</strong>medium within <strong>the</strong> loop. A single embryo waspipetted onto <strong>the</strong> film. The cryoloop containing <strong>the</strong>embryo was <strong>the</strong>n immediately plunged into <strong>the</strong>cryovial, which was already submerged underliquid nitrogen. The cryovial lid was sealed underliquid nitrogen in one motion. The intervalbetween contact with concentrated cryoprotectantand plunging was 20–30 s. Embryos were stored in54