Surviv land viability of vitrified in vitro and in vivo produced ovine blastocysts

ELSEVIER

SURVIVP L AND VIABILITY

OF VITRIFIED IN VITRO AND IN VIVO PRODUCED BLASTOCYSTS

OVINE

M. Dattena,la G. Ptak,lv2 P. Loi ’ and P. Cappair ‘Istituto Zootecnico e Caseario per la Sardegna, Sassari, Italy 2Department of Animal Reproduction, University of Agriculture, Krakow, Poland Received for publication: Accepted:

May 7, 1999 November 3, 1999

ABSTRACT Ovine blastocysts were produced by maturation, fertilization and in vitro culture (IVM/lVF/IVC) of oocytes from slaughteredadult and prepubertalewes and collection from superovulatedand inseminatedadult animals.Dulbecco’sPBS supplementedwith 0.3 mM Na Pyruvate and 20% FCS was used as the basic cryopreservationsolution. The embryoswere exposedto the vitrification solutionas follows: 10% glycerol (G) for 5 min, then 10% G +20% ethyleneglycol (EG) for 5 min. Embryoswere placedinto 25% G + 25% EG in the center of 0.25- mL strawsand plungedimmediatelyinto LN2 . Warming was doneby placing the straws into a water bath at 37°C for 20 set, and their contentswere expelled into a 0.5 M sucrose solutionfor 3 min; the embryoswerethen transferredinto 0.25 M and 0.125M sucrosesolution for 3 min each.Warmedblastocystswere transferredto the culture mediumfor 24 h. Survival wasdefinedasthe re-expansionof the blastocoele.All surviving blastocystswere transferredto synchronizedrecipient ewes,and the pregnancywas allowed to go to term. Of 68 vitrified in vitro producedblastocysts,46 re-expanded(67.6%) and 10 lambswere born (14.7%). From the 62 in vivo derived and vitrified embryos, 52 re-expanded(83.8%) and 39 lambswere born (62.9%). The lambingrate of in vitro producedfresh transfer embryoswas 40% (20 lambs/50 blastocyststransferred),andof the 32 in vivo derived blastocystsand transferredfresh,26 lambs were born (81.2%). The resultsindicate that in vitro producedembryos can be successfully cryopreservedby vitrification. 0 2000

by Elsevier

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Inc.

Key words: sheep,embryo,in vitro production,in vivo-derived, vitrification Acknowledgments The authorsthank Mr. G. Camoglioand Mr. A. Pintadufor animalmanagementand Mr. F. Chessafor technicalassistance during surgery. aCorrespondence andreprint requests:Istituto Zootecnicoe Casearioper la Sardegna,Servizio RiproduzioneAnimale. 07040Olmedo(SS), Italy, E-mail: [email protected] Theriogenology Q 2000 Elsevier

63:161 I-1619,200o Science Inc.

0093-691WOO/$-see front PII SOO93-691X(00)00293-4

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INTRODUCTION The first lamb producedby IVF and IVC of IVM oocyteswas in 1991(5, 25). Major advanceshave sincebeenmadein IVM, IVF andNC of adult and prepubertaloocytes.Several groupshave reportedthe birth of lambsfollowing the useof thesetechniques(3, 4, 10, 22, 28, 36, 40). In vitro embryo production is supportedby cryopreservationtechnology. Among the different techniquesof cryopreservation(8, 32, 44), controlled slow freezing and vitrification, which involves the additionof higherconcentrationsof cryoprotectantandvery rapid cooling (8) arethe mostwidely used.In the pastdecadevitrification hasbeenusedin cowsandsheep(2, 13, 18, 21, 24, 41), and it is now regardedas a potential alternative to the traditional slow freezing method (12, 13, 14, 31, 42). Cryopreservationof in vitro produced sheepembryosby slow freezing and vitrification has already been reported (15, 30); however, a comparisonof the freezability of in vitro- andin vivo-produced sheepembryosis lacking. The potential of vitrified embryosto developinto viable lambsalsoneedsto be investigated. In our presentwork we comparethe post transfersurvival of in vitro-produced vitrified and fresh blastocysts,both from adult and prepubertalanimals,and of in vivo-derived embryos from superovulatedewes. MATERIALS AND METHODS Except where otherwiseindicated,all chemicalswere obtainedfrom Sigma Chemicals Company(St. Louis, MO, USA). In Vitro Embryo Production Ovaries were collected immediatelyafter slaughterand transportedto the laboratory in salineat approximately 35°C within 1 to 2 h. Oocytes were obtainedby aspirationof follicles using a 20-gaugeneedlefitted with a 2-mL syringe. Follicular oocytes covered by at least 2 layers of granulosacells and with an evenly granulatedcytoplasmwere selectedfor IVM. The mediumusedfor maturationwasbicarbonate-bufferedTCM-199 with the osmolarityadjustedto 275 mOsm and glutaminepresentat a concentrationof 2 mM. The maturation mediumwas enrichedwith 10% fetal bovine serum(FBS), 5 ng/mI.. FSH (Ovagen, ICP, Auckland, New Zealand)5 ng/mL LH, 1 pg/mL estradiol,0.3 mM sodiumpiruvate and 100pM cysteamine.The oocytes were incubated in 400 nL of medium in 4-well dishes(Nunc, Nunclon, Denmark) coveredwith mineraloil. In vitro maturationconditionswere 5% CO2in humidified air at 39°C for 24 h. Following maturation, the oocytes were partially denudedof granulosacells by gentle pipetting in Hepes-TCM-199with 300 I.U./mL hyaluronidase.Freshsemenfrom a Sardabreed ram of proven fertility wasusedthroughoutthe experiment.Collectedejaculatewaskept at room temperaturefor up to 2 h, then washedin Hepes-SOFand centrifugedtwice at 200 x g for 5 min and subsequentlyaddeddirectly to the fertilization medium.The mediumusedwas bicarbonatebuffered synthetic oviduct fluid (SOF), as originally describedby Tervit et al. (37) enrichedwith 20% (v/v) heatinactivatedestroussheepserum,2.9 mM Ca lactateand 16 nM isoproterenol(25).

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Fertilization wasconductedin 50-uL dropswith 1 x 1O6sperm/ml andmaximum15 oocytesper drop, at 39°C with 5% CO2in humidifiedair for 20 h. The presumptivezygotes were allocatedto 2O+L culture drops (4 to 5 embryos/drop) consistingof SOF supplementedwith 2% (v/v) BME-essentialamino acids, 1% (v/v) MEMnonessentialamino acids, 1 mM glutamineand 8 mg/mL fatty acid free BSA. The incubation conditionswere 5% 02, 5% CO2,90% Nz, 5% charcoalstrippedFBS addedto the mediumon the third andfifthday of culture (Day 0 the day of fertilization; 39). Culturewascontinueduntil 6 to 7 d post fertilization, then embryoswhich developedat leastto expandedblastocystswere transferredto synchronizedrecipientsor werevitrified. In Vivo Derived Embryo The estrouscycle of 16 Sardaeweswassynchronizedby insertionof intravaginalsponges containing40 mg fluorogestoneacetate(Intervet, NL) for 14 d. The day before removal of the spongesthe ewes were superovulatedwith 16 mg FSH (FSH-p; Pluset, Serono, Italy) administeredintramuscularlyevery 12h in 4 decreasingdoses(6, 5, 3 and2 mg). The eweswere inseminatedwith fresh semenfrom a fertile ram 48 h after spongeremoval by intrauterine insemination.Embryos were collected from the donors under general anesthesia(Pentothal sodium,Gellini, Italy) 7 d from the start of estrus.Each uterine horn was flushedusinga Foley catheter as describedby Tervit and Havick (34). The collection medium was TCM 199 supplementedwith Hepesand 4 mg/mL BSA. The embryoswere classifiedaccordingto their stageof development.Of the 112embryoscollected94 were at the expandedblastocyststageand only thosewere subjectedto immediatefreshtransferor to vitrification. Four replicateswere madefor eachsourceof embryos. Vitrification ProceduresandWarming The vitrification procedureemployedthroughoutthis experimentis basedon the method originally designedby Yang et al. (46) for in vivo cow embryos,and previously usedin our laboratory for in vivo-derived sheepembryos (21). Briefly, all vitrification solutions were preparedusing PBS supplementedwith 0.3 mM sodiumpyruvate, 3.3 mM glucoseand 20% FBS. Expandedovine blastocystswere exposedto the vitrification solutionat 20” C accordingto the following procedure:glycerol 10% (G) for 5 min, then to glycerol 10%plus ethyleneglycol 20% (EG) for 5 min before being transferredinto a 25-uL column of 25% glycerol and 25% ethylene glycol, then loaded into the center of 0.25-mL plastic inseminationstraws (IVM, L’Aigle, France)usinga fine glasscapillary pipette. In the straws,the embryosand vitrification solutionwere separatedby 2 air bubblesfrom columnsof 0.5 M sucrosesolution(90 uL) each. After heat-sealingthe strawswere plungedimmediatelyinto LN2. The embryoswere warmedby holdingthe strawsfor 6 set in air andthen agitatingthem in a water bath at 37°C for at least 15 sec.The contentsof eachstraw were emptiedinto a Petri dishandstirredgently to facilitate mixing of the 2 solutions.The embryoswerepicked up with a glasscapillary pipette and transferredto 0.25 M and 0.125M sucrosesolutionfor 3 min eachto allow for removal of intracellular cryoprotectant.Then they were held for 24 h in TCM 199 enrichedwith 20% FCS in humidified air containing5% CO2 at 39°C to permit re-expansionof the blastocoele.

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Transplantationof IVP andND-Vitrified Embryos Only the re-expandedblastocystsweretransferred,in pairs,to recipientewes7 d after the onsetof natural estrus.Pregnanciesconfirmed by ultrasonographyat 40 d were allowed to be carriedto term. Transplantationof IVP andIVD-Fresh Embryos After 6 to 7 d of culture only blastocystswere transferredin pairsinto recipientewes7 d after the onsetof the estrus.The in vivo derived blastocystsweretransferreddirectly in pairs2 h after flushingto synchronizedrecipientewes.Pregnanciesconfirmedby ultrasonographyat 40 d wereallowedto be carriedto term. StatisticalAnalysis Comparisonbetweengroupswas performedby the Chi-squaretest (SASSTAT User’s Guide, 6.03 edition, SAS Institute Inc., Cary, NC). RESULTS A total of 450 oocytes,recoveredfrom slaughterhouse-derived adult ewe ovaries,were in vitro matured,and of these335 cleaved (74.4%). Of the 118 (35.2%) blastocystsobtained,50 were transferred fresh and 68 were vitrified. A total of 390 oocytes recovered from slaughterhouse-derived prepubertalewe ovarieswere in vitro maturedand 281 of thesecleaved (72.0%). Of the 281 cleavedembryos,63 (22.4%) developedinto blastocysts.Twenty embryos weretransferredfreshand 43 werevitrified. The survival of vitrified embryoswasassessed by re-expansionof the blastocoeleduring 24 h of culture. There was a significant difference (P
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DISCUSSION Vitrification offers severaladvantagesin embryo storagedue to its simplicity and low cost. Moreover, vitrification of in vitro-produced embryos and in vivo-derived embryos of species,particularly sensitiveto freezing suchas pig embryosyields resultssimilar to and in somecaseshigherthan conventionalfreezing (6, 11, 14, 31,42). It is well known that in vitro produced embryos have an increasedsensitivity to temperaturesbelow 14°C (24). Most of the standardmethodsof cryopreservationrequirethat embryosbe cooled slowly to below physiological temperatures,and this can causeserious damagein in vitro producedembryos.This effect could possiblybe minimized if embryoswere cooledrapidly by direct immersionin LN2, asin vitrification (6, 11, 24). Thus, embryo survival of in vitro producedembryosis affectedby the choiceof cryopreservationtechnique(vitrification vs slowfreezing). Moreover, the type of the cryoprotectant,its concentration,lengthof exposure, warming temperature, concentration of nonpermeable cryoprotectant, and the protein compositionof the freezing mediumalso affect the survival of embryos.Additionally, nearly every laboratoryhasa different cryopreservationprotocol, makingresultsdifficult to compare.A number of studieshave shownthat a high survival rate of cryopreservedin vitro produced embryosnecessitateimprovementin the techniquesusedfor in vitro maturationand culture, as well as in cryopreservationmethods(9, 16, 24). For example,the type of protein during in vitro culture canaffect freezing resistance,ashasbeenreportedrecently (6, 20, 23). In our presentstudy lambing results of the in vitro producedvitrified embryoswere higherthan in earlier reports(15, 30). The overall lambingrate of vitrified/re-expandedembryos was lower than that of fresh embryos,although it was not significantly different due to the limited numberof observations.The lambingrate calculatedasnumberof lambsborn per total number of vitrified embryoswas even lower. To our knowledge,there are no reports where pregnancyand lambingof in vitro produced-vitrified and fresh embryoswere compared.Most previous reports on cow embryosproducedin vitro do not show differencesin calving rates betweenrecipientsbearingfreshand cryopreservedembryos(1, 18, 3l), but one study does(43). However, there are fundamentaldifferencesin cellular structurebetweenin vivo- and in vitroderived embryos,with the in vitro-produced embryosbeing the more sensitiveto freezing as reportedby Leibo and Loskutoff (13). Theseworkersdescribethe difference in buoyant density between in vivo and in vitro embryosdue to the different ratio of lipids and proteins, with characteristicfloating of the embryo when immersedin a sucrosesolution. Other studieshave reported that there is varying susceptibilityof the zonae to pronasedigestion.This and other conditionssignificantly affect sensitivity to freezing of in vitro-producedembryos(16,24). The re-expansionrate of in vitro producedvitrified blastocystsfrom prepubertaloocytes is significantly lower than of in vitro producedvitrified blastocystsfrom adult sheepoocytes, suggestingthat embryosfrom prepubertaloocytesmay be moresensitiveto cryopreservation,but that thosewhich reachthe re-expansionstagehave the capacity to developto term. As expected, the rates of re-expansion,pregnancy and lambing, were significantly lower in the in vitro producedvitrified blastocysts,both from adult and prepubertalanimals,than in in vivo derived vitrified blastocysts(P
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our study yielded resultscomparableto thoseof other reports(33, 38). Yet, the lambingratesof in vivo derived sheepembryostransferredfresh or after cryopreservationwere similaror higher than thosereportedby other workers (2, 19, 27, 29). A few works have describeddifferences between in vitro produced and in vivo derived embryosfrom domesticruminants(38, 45). Synchrony of development, cell numbers, gap junction, cytoplasmic appearance,alterated metabolic profiles, protein synthesisand gene expressionare the most commonly occurring differencesamongthese2 types of embryos.Although it wasoutsidethe scopeof this study to describeall of the existing differences,eachuniquely contributesto the quality and consequent survival of embryos(7, 38,39,45) andto the success of their freezability (13, 16,20,35). Basedon current results, overall efficiency of vitrified embryos is encouraging.The conventionalidea that in vitro producedembryoshave a lower survival rate following transfer than in vivo-derived embryosis basedmainly on data in which embryosfrom only 1 sourcewere transferred.This is the first time that viability after transferof fresh andvitrified sheepembryos, both in vitro producedand in vivo derived,is reported. Theseresults indicate a gap betweenin vitro and in vivo viability between embryos transferredvitrified or fresh.Optimization of the cryopreservationtechnologyis neededto reduce chilling injury, crystal formation, and toxic and osmotic stressfor all sourcesof embryos. Improving the culture systemwould improveresistanceat freezing of embryosproducedin vitro. REFERENCES 1. AgcaY, Monson RL, Northey DL, Abas Mazni 0, Rutledge JJ. Post-thaw survival and pregnancyrates of in vitro producedbovine embryosafter vitrification. Theriogenology 1994;41:154abstr. Ali J, Shelton JN. Successfulvitrification of day-6 sheepembryos. J Reprod Fertil 1993;99:65-70. Brown BW, Radziewic T. Production of sheepembryos in vitro and developmentof progenyfollowing singleandtwin embryotransfers.Theriogenology1998;49:1525-l536. Cognii!Y. Stateof the art in sheep-goat embryotransfer.Theriogenology1999;51:105-l16. CzlonkowskaM, Eysymont U, Guszkiewicz A, KossakowskiM, Dziak J. Birth of lamb after in vitro maturation, fertilisation, and co-culture with oviductal cells. Mol Rep Dev 1991;30:34-38. 6. DinnyesA, CarolanC, LonerganP, MassipA, Mermillod P. Survival of frozen or vitrified bovine blastocysts produced in vitro in synthetic oviduct fluid. Theriogenology 1996;46:1425-1439. 7. Dorland M, Gardner DK, Trounson AO. Serum in synthetic oviduct fluid causes mithocondrialdegenerationin ovine embryos.J ReprodFertil 1994;102(Abstr Ser 13):70 abstr. 8. Fahy GM, MacFarlaneDR, Angel1 CA, Meryman HT. Vitrification as an approachto cryopreservation.Cryobiology 1984;21:407-426. 9. Greve T, Avery B, CallesenH. Viability of in vivo and in vitro producedbovine embryos. ReprodDom Anim 1993;28:164-169. 10. Holm P, Walker SK, PetersonBA, AshmanRJ, SeamarkRF. In vitro vs in vivo culture of nvine NM-NF ova: effect on lambing.Theriogenologv1994;41:217abstr.

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29. SongsasenN, Buckrell BC, Plante C, Leibo SP. In vitro and in vivo survival of cryopreservedsheepembryos.Cryobiology 1995;32:78-91. 30. SongsasenN, Walmsley S, Pollard JW, Martin0 A, Buckrell BC, Leibo SP. Lambs producedfrom cryopreservedsheepembryosderived by in vitro fertilization of aspirated oocytes.CanJ Anim Sci 1996;76:465-467. 31. TachikawaS, Otoi T, Kondo S, Machida T, Kasay M. Successfulvitrification of bovine blastocystsderived by in vitro maturationand fertilization. Mol ReprodDev 1993;34:266271. 32. TakahashiY, KanagawaH. Effect of equilibrationperiodon the viability of frozen-thawed mousemorulaeafter rapid freezing. Mol ReprodDev 1990;26 (2): 1OS-110. 33. Tervit HR. Laparoscopy/laparotomy oocyte recovery andjuvenile breeding.Anim Reprod Sci 1996;42: 227-238.. 34. Tervit HR, Havik PG. A modified techniquefor flushing ova from the sheeputerus.NZ Vet J 1976;24:138-140. 35. Tervit HR, Pugh PA, McGowan LT, Bells ACS, Wells LW. The freezability of sheep embryos is affected by culture system and source (in vivo- or in vitro-derived). Theriogenology1994;41:315 abstr. 36. Tervit HR, Smith JF, McGowan LW, PoughPA. Birth of lambsfrom embryosproducedin vitro following laparoscopicrecovery of follicular oocytes. Proc Aust Sot Reprod Biol 1995;27:68abstr 37. Tervit HR, Whittingh DG, Rowson LEA. Successfulculture of sheepand cattle ova. J ReprodFertil 1972;30:493-497. 38. ThompsonJG. Comparisonbetweenin vivo-derived and in vitro-producedpre-elongation embryosfrom domesticruminants.ReprodFertil Dev 1997;9:341-354. 39. ThompsonJG, Allen NW, McGowan LT, Bell ACS, LambertMG, Tervit HR. Effect of delayed supplementationof fetal calf serum to culture medium on bovine embryos developmentin vitro and following transfer.Theriogenology1998;49:1239-1249. 40. ThompsonJG, Bell ACS, McMillan WH, AJ Peterson,Tervit HR. Factors affecting in vitro developmentand post-transfersurvival of cultured sheepembryos.Theriogenology 1994;41:316abstr. 41. Vajta G, Holm P, Greve T, CallesenH. Overall efficiency of in vitro embryo production andvitrification in cattle. Theriogenology1996;45:683-689. 42. Vajta G, Holm P, KuwayamaM, Booth PJ, JacobsenH, Greve T, CallesenH. Open-Pulled Straw (OPS) vitrification: a new way to reducecryoinjuries of bovine ova and embryos. Mol ReprodDev 1998;51:53-58. 43. Van Soom A, Mjtien P, Van VlaenderenI, Van Den Branden J, MahmoudzadehAR, DeKruif A. Birth of double-muscled Belgianblue calvesafter transferof in vitro-produced embryosinto dairy cattle. Theriogenology1994;41:855-867. 44. Whittingam DG. Survival of mouse embryos after freezing and thawing. Nature 1971;233:125-126. 45. Wright RW Jr, Ellington J. Morphological and physiologicaldifferencesbetweenin vivoand in vitro-produced preimplantationembryos from livestock species.Theriogenology 1995;44:1167-1189. 46. Yang NS, Lu KH, GordonI, PolgeC. Vitrification of bovine blastocystsproducedin vitro. Theriogenology1992;37:326abstr.