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

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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
Equine Embryo TransferCOMPARISON OF GLYCEROL AND ETHYLENEGLYCOL IN EQUINE EMBRYO FREEZING USINGCONFOCAL MICROSCOPY, DAPI-STAINING ANDNONSURGICAL TRANSFERM. Huhtinen, A. Sjöholm and J. Paranko*MTT, Equines, Ypäjä, Finland; *Department <strong>of</strong> Biology, Laboratory <strong>of</strong> Animal Physiology, University <strong>of</strong>Turku, FinlandINTRODUCTIONAfter reaching a certain diameter anddevelopmental stage, equine embryos are severelydamaged during freezing. Several cryoprotectantshave been tested in an attempt to overcome thisproblem, but so far, only glycerol hasgiven reasonable results in conventionalcryopreservation procedures (for review see Seidel1996). In <strong>the</strong>ory, <strong>the</strong> higher permeability <strong>of</strong>ethylene glycol versus glycerol (Pfaff et al. 1993)would favour <strong>the</strong> use <strong>of</strong> <strong>the</strong> former. The availability<strong>of</strong> reliable techniques to study damages induced byfreezing and thawing <strong>of</strong> embryos have beenlimited. One such tool could be confocalfluorescence microscopy because it enables a 3-dimensional analysis <strong>of</strong> o<strong>the</strong>rwise difficult intactbiological specimens, like fluorescently labelledmulticellular embryos. The aim <strong>of</strong> <strong>the</strong> present workwas to compare cryodamage in equine embryosafter freezing in glycerol or ethylene glycol usingconfocal microscopy, DAPI-staining, andnonsurgical transfer.MATERIALS AND METHODSThirty-seven embryos, 140–190 µm in diameter,were frozen in a programmable freezer afteraddition <strong>of</strong> <strong>the</strong> selected cryoprotectant. Glyceroland 50 mM glutamine were added in 4 steps for18 embryos. Nineteen embryos were placed into1.5 M ethylene glycol in Emcare. All embryoswere thawed by holding <strong>the</strong> straws in <strong>the</strong> air for10 s and <strong>the</strong>n in a water bath at 30ºC for 20 s. In<strong>the</strong> glycerol group, <strong>the</strong> cryoprotectant wasremoved in six steps with sucrose (Huhtinen et al.1997). Half <strong>of</strong> <strong>the</strong> embryos in each treatmentgroup were stained with DAPI (1 µg/ml) for 15min in room temperature, washed and transferrednonsurgically to <strong>the</strong> uteri <strong>of</strong> recipient mares thathad ovulated four days before transfer.The remaining half were fixed in 3.7%formaldehyde for 15 min in room temperature.After three washes in PBS, <strong>the</strong>y were extractedwith <strong>the</strong> solution <strong>of</strong> 0.1% Triton X-100 inPBS for 5 min. After 3 washes in PBS, <strong>the</strong>embryos were treated with RNAse (1 mg/ml) inPBS for 15 min. After short washes in PBS, <strong>the</strong>embryos were incubated for 15 min in PBScontaining 1% BSA. Then <strong>the</strong> embryos wereincubated in <strong>the</strong> presence <strong>of</strong> Oregon Greed 514phalloidin and propidium iodide (0.5 µg/ml) for30 min in room temperature. After 3 washes inPBS, <strong>the</strong> embryos were mounted on a slide in 1%agar and analysed with Leica TCS NT confocalmicroscope.TABLE 1: Number/percentage <strong>of</strong> DAPI-stained cells in frozen-thawed embryos before transferEmbryo number1 2 3 4 5 6 7 8 9 10Gly 0* 1 6* 10 10* 11 25 20%* 30% -EG 4* 10* 14 15 30 35 35 50 30%* 40%* = The embryo continued its development after transfer52
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Equine Embryo TransferChromatin con
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