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

Havemeyer Foundation Monograph Series No. 3NOVEL METHODS OF EMBRYO CRYOPRESERVATIONS. P. LeiboAudubon Center for Research <strong>of</strong> Endangered Species, Department <strong>of</strong> Biological Sciences, University<strong>of</strong> New Orleans, New Orleans, Louisiana, USAThe capability to cryopreserve mammalian embryosis a powerful adjunct to methods <strong>of</strong> assistedreproduction. Tens <strong>of</strong> thousands <strong>of</strong> mice andhundreds <strong>of</strong> thousands <strong>of</strong> cattle have been producedfrom cryopreserved embryos. In contrast, onlyabout 50 equine pregnancies and fewer than 10 foalsresulting from cryopreserved conceptuses have beenreported in <strong>the</strong> literature. Because <strong>of</strong> innumerableinvestigations <strong>of</strong> <strong>the</strong> cryobiology <strong>of</strong> embryos,especially those <strong>of</strong> mice and cattle, considerablemechanistic understanding has been derived <strong>of</strong> <strong>the</strong>factors that determine ultimate survival and fulltermdevelopment <strong>of</strong> cryopreserved embryos.However, <strong>the</strong>re have been many fewer studies <strong>of</strong> <strong>the</strong>basic cryobiological aspects <strong>of</strong> equine embryos thatmay influence <strong>the</strong>ir response to freezing andthawing. Any such investigation must take intoaccount unique features <strong>of</strong> <strong>the</strong> equine conceptus,and also <strong>the</strong> unusual reproductive biology <strong>of</strong> <strong>the</strong>mare compared to <strong>the</strong> female <strong>of</strong> many o<strong>the</strong>r species.Cryopreservation <strong>of</strong> embryos <strong>of</strong> all speciesconsists <strong>of</strong> <strong>the</strong> following steps:1. Exposure to molar concentrations <strong>of</strong> one or morecryoprotective additives (CPAs);2. Cooling to sub-zero temperatures underconditions that result in efflux <strong>of</strong> virtually allintracellular water from <strong>the</strong> embryos;3. Immersion in liquid nitrogen (LN 2 ) at -196°C,permitting unlimited storage with no loss <strong>of</strong>biological function;4. Warming to physiological temperatures;5. Removal <strong>of</strong> <strong>the</strong> CPAs from <strong>the</strong> embryos.Cryopreservation is a unidirectional process;consequently, all <strong>the</strong> steps <strong>of</strong> <strong>the</strong> sequence must beperformed properly if an embryo is to survivecryopreservation. But each <strong>of</strong> <strong>the</strong>se steps has <strong>the</strong>potential to damage or destroy mammalianembryos. For example, common CPAs are glycerol,dimethyl sulphoxide, ethylene or propylene glycol.Concentrated solutions (10–50% by volume) <strong>of</strong>those CPAs may damage embryos, ei<strong>the</strong>r by directtoxic effects or by osmotic shock, during <strong>the</strong> initialexposure or during its removal. Very few studies <strong>of</strong><strong>the</strong> effects <strong>of</strong> CPAs on equine embryos have beenconducted. Ano<strong>the</strong>r factor that may affect embryodevelopment is exposure to non-physiologicaltemperatures. Although embryos may ‘tolerate’exposure to temperatures near or below 0°C, it isbecoming increasingly evident that embryos mayundergo subtle injury caused by chilling. There aresignificant differences in chilling sensitivity amongembryos <strong>of</strong> different species, and among variousdevelopmental stages <strong>of</strong> a given species. Little isknown about <strong>the</strong> susceptibility <strong>of</strong> equine embryos tochilling injury. A third factor known to have a majoreffect on embryo survival is <strong>the</strong> rate at whichsamples are cooled from ~0°C to sub-zerotemperatures below -30°C. Only one very briefstudy <strong>of</strong> this variable in <strong>the</strong> horse conceptus hasbeen reported. Most investigations have used asingle cooling rate <strong>of</strong> 0.3°C/min to freeze equineembryos; this may or may not be <strong>the</strong> optimum rate.Yet ano<strong>the</strong>r potentially damaging event may occurduring removal <strong>of</strong> <strong>the</strong> CPA from <strong>the</strong> embryo after itscryopreservation. The effect <strong>of</strong> CPA removal onembryo survival depends on <strong>the</strong> permeability <strong>of</strong> thatspecific embryonic stage to <strong>the</strong> CPA itself and <strong>the</strong>temperature coefficient <strong>of</strong> permeability. There aresignificant differences among species with respectto embryo permeability; little is known about <strong>the</strong>relationship between permeability to various CPAsand temperature <strong>of</strong> <strong>the</strong> equine conceptus.In summary, systematic studies <strong>of</strong>cryobiological variables known to affect survival <strong>of</strong>embryos <strong>of</strong> o<strong>the</strong>r mammalian species, as well asstudies that allow for exceptional characteristics <strong>of</strong><strong>the</strong> equine conceptus may yield improved methodsfor <strong>the</strong>ir cryopreservation.51