Theriogenology 41:

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1 Theriogenology 41: VIABILITY OF FROZEN-THAWED BOVINE IVM/IVF EMBRYOS IN RELATION TO AGING USING VARIOUS CRYOPROTECTANTS M. Tkgi 1 IT. Otoi 2, A. Boediono 1, S. Sh 1 nd T. Suzuki! 1 United Grdute School of Veterinry Science Ymguchi University, Ymguchi 753, Jpn nd 2 Tokushim Prefecturl Beef Cttle nd Swine Experiment Sttion, Ann, Tokushim 774, Jpn Received for publiction: My 7~ 1993 Accepted: November 4~ 1993 ABSTRACT Bovine IVF embryos developed on Dys 7, 8 nd 9 were equilibrted with 1. 6 M propylene glycol (PG), 1. 8 M ethylene glycol (EG), 1. 1 M diethylene glycol (DEG) or 1. 3 M ethylene glycol monomethyl ether (EME) for 10 to 20 min in modified phosphte buffered sline (mpbs) supplemented with 10% superovulted cow serum. The embryos were loded into ml plstic strws nd were plced directly into O"C lcohol bth chmber nd held for 2 min. They were cooled from O"C to -5.5"C t 1"C /min nd then seeded, followed by 10-min holding period t "C. The strws were then cooled to -30"C t 0. 3"C /min before plunging into liquid nitrogen. Embryos were thwed nd plced directly into the culture medium nd wshed 3 times. The survivl rtes of the Dy-9 embryos bsed on reppernce of blstocoele, expnsion, nd htching fter 48 h of post-thw culture were significntly lower (P<0.01) thn those of the Dy-7 nd 8 embryos, in ll of the cryoprotectnts tested. On the other hnd, while the reppernce of blstocoele nd expnsion of blstocysts fter 48 h of post-thw culture were not significntly different mong ech cryoprotectnt, the percentge of htching blstocysts were significntly different between DEG nd EME (P<0.05) 1 between DEG nd EG (P<0.01) nd between PG nd EG (P<0.05). These findings demonstrte tht the ge of the embryo (Dy 7 nd 8) is very importnt for the successful freezing of IVF bovine embryos. Also, s to the htching rtes, EME nd EG re superior s cryoprotectnts thn the other 2 cryoprotectnts tested. Key words: bovine, IVF embryo, freezing, cryoprotectnts INTRODUCTION Recently, dvnces in embryo trnsfer technology hve enbled us to produce bovine embryos through the fertiliztion nd development of in vitro mtured oocytes derived from slughtered cttle. Blstocysts thus produced hve the bility to develop into live clves fter trnsfer into recipient cows (5, 6). Therefore, Acknowledgments We wish to thnk Dr. H. Hmnk, Boron Lbortory, for the gift of cryoprotectnts; Dr. K. Mtsuok, Ymguchi Prefecturl Zootechnicl Experiment Sttion, for the gift of superovulted cow serum; Dr. D. J. Ambrose, University of British Columbi, for his helpful evlutio'n of the mnuscript. Copyright 1994 Butterworth-Heinemnn

2 916 Theriogenology the cryopreservtion of these embryos will hve n importnt role in the prcticl ppliction of this technique. However, there hve been only few preliminry reports on the cryopreservtion of bovine embryos produced in vitro (4, 7, 16). The survivl rtes of frozen-thwed IVF bovine embryos re ffected by the qulity or ge of the embryos, cryoprotectnt used, the cooling rtes during freezing, nd the method of equilibrtion or removl of the cryoprotectnt (4,7,16-18). Mukojim et l. (11) reported tht the pregnncy rte of the recipient receiving fresh IVF bovine embryos ws not ffected by embryo ge. However, Tchikw et l ( 16) reported tht the in vitro developmentl rte of fresh IVF blstocyst& obtined on Dy 9 fter insemintion ws lower thn tht obtined on Dy 7 or Dy 8. In frozen-thwed bovine embryos, the cryoprotectnt must be diluted or removed before culture or trnsfer into the uterine horn. Until now, stepwise method hs been used to remove the cryoprotectnt (1,19,20). Direct trnsfer methods (one-step or two-step method) hve lso been described for in vivo bovine embryos (8, 12-14). Recently, some investigtors hve reported direct trnsfer methods without dilution or removl of the cryoprotectnt, with or without sucrose, using glycerol (9), propylene glycol ( 15) nd ethylene glycol (17,18) In this study, we exmined whether embryo ge fter insemintion ffected the survivl rte of the post-thw IVF embryos frozen in vrious cryoprotectnts (ethylene glycol, diethylene glycol, propylene glycol nd ethylene glycol monomethyl ether) nd thwed by direct trnsfer methods. MATERIALS AND METHODS In Vitro Fertiliztion of Ovrin Oocytes Cumulus-oocyte complexes (COCs) were spirted from smll ntrl follicles (1 to 5 mm in dimeter) in bovine ovries obtined from locl slughterhouse. After collection, only oocytes with n intct, unexpnded cumulus oophorus were wshed twice with the mturtion medium (25 mm-hepes TCM-199 with Ergle's slts; Gibco, NY, USA) supplemented with 5% serum collected from superovulted cows on Dy 7 of their cycle (SCS; 10), AU/ml FSH (Denk, Kwski, Jpn) nd 50 flg/ml of Gentmicin sulfte (Sigm, St.Louis, USA). Therefter the COCs were plced into the mturtion medium nd cultured for 21 to 22 h t 38.5t with 5% C0 2 in ir. Frozen semen from single bull ws used. The semen ws wshed twice with BO medium (2), without bovine serum lbumin, nd supplemented with 5mM-cffeine, by centrifugtion, t 500 x g for 5 min. The sperm pellet ws resuspended in BO medium contining bovine serum lbumin (5 mgjml, Sigm, St.Louis, USA) nd 2.5 mm cffeine for sperm concentrtion of 5x 10 6 /ml. A 100-!ll liquot of the sperm suspension ws incubted for 1 h t 38. 5t with 5% C0 2 in ir prior to insemintion. Insemintion nd subsequent culture were performed ccording

3 Theriogenology 917 to the method reported by Kjihr et l (6). The COCs cultured for 2 1 to LL n were trnsferred to ech sperm microdroplet for insemintion. After the incubtion of spermtozo nd oocytes for 5 h, ech oocyte ws trnsferred to culture medium (25 mm Hepes TCM-199 supplemented with 5% of SCS 5JJg/ml insulin (Sigm, St. Louis, uuft.j, nd 50 i-jl/ml gentmicin sulfte i 18} nd cultured for up to 9 d fter insemintion t 38.5t in 5% C0 2 in ir. Morphologiclly norml blstocyst& nd expnded blstocysts which uou developed to tht ;::.L.ctye on Dys 7, 8, 9 following in vitro fertiliztion were used in this experiment. Freezing-Thwing nd Culture Embryos were suspended in Dulbecco's phosphte buffered sline (D-PBS; Gibco, NY, USA) supplemented with 10% SCS nd 1.6 M propylene glycol (PG; Wko Pure Chemicl, Osk, Jpn) 1. 8 M ethylene glycol(eg; Wke Pure Chemicl, Osk, Jpn), 1.1 M diethylene glycol (DEG; Boron Lbortory, Sitm, Jpn) or 1.3 M ethylene glycol monomethyl ether (EME; Boron Lbortory, Sitm, Jpn). The cryoprotectnts were dded directly t room temperture (20 to 250C ). After 10 to 20 min equilibrtion, the embryos were loded into 0.25-ml plstic strws, plced directly into Ot lcohol bth chmber, nd held for 2 min. Then the embryos were cooled from Ot to -5.5t t 1t /min nd seeded t -5.5t. After seeding, the strws were held for 10 min t -5.5t, nd cooled t rte of 0.3t /min to -30t. Finlly, the strws were plunged nd stored in LN 2 Embryos were thwed by plcing the strws in 30t wter bth, nd the contents drined into sterile petri dish. The embryos were then plced into the culture medium for rehydrtion nd wshed 3 times. Embryos were cultured on feeder lyers of bovine cumulus cells in TCM-199 supplemented with 5% SCS nd 5JJg/ml insulin under 38.5t in 5% co?. in ir. Embryos were evluted microscopiclly t 24 h intervls for 48 h. Survivl rtes were ssessed t 48 h culture, by 3 prmeters: 1) re-ppernce of the blstocoele 2) ttinment of fully expnded stge (including the htching nd htched blstocyst) nd 3) bility to htch in vitro. The effect of the cryoprotectnt on subsequent development into bove criterions for ech qe qroup ws tested by n nlysis of vrince (ANOVA). A probci:'bili ty of ws considered significnt. RESULTS Tble 1 shows the survivl rtes of in vitro produced bovine embryos frozen in vrious cryoprotectnts, ssessed by reppernce of the blstocoele fter 48 h of culture. The survivl rtes in the Dy-9 embryos were significntly lower thn those in the Dy-7 nd 8 embryos mong 4 cryoprotectnts (P<0.01).

4 918 Theriogenology Tble 1. Survivl rtes (%) of in vitro produced bovine blstocysts frozen with vrious cryoprotectnts. Survivl rtes were ssessed by re-ppernce of the blstocoele fter 48 hours of culture. Cryoprotectnt Age of embryo Dy 7 Dy Dy 9- AN OVA EME 94.4 (71) 76.3 (38) 65.2 (23) E G DEG p G (68) 81.7(60) 81.4 (43) (36) (32) 82.5 (40) (43) 29.2 (24) 55.0 (20) ANOVA b Vlues in rows nd columns with no letters in common re significntly different (P<0.01). EME~Ethylene glycol monomethyl ether; EG~Ethylene glycol; DEG~Diethylene glycol; PG~Propylene glycol. Figures in prenthesis indicte number of embryos. Tble 2 shows the survivl rtes bsed on ttinment of fully expnded stge fter 48 h culture. The rte of expnsion of blstocysts in the Dy-9 embryos ws significntly lower thn those in the Dy-7 nd 8 embryos mong 4 cryoprotectnts (P<0.01). Tble 2. Survivl rtes (%) of in vitro produced bovine blstocysts frozen with vrious cryoprotectnts. Survivl rtes were ssessed by ttinment of fully expnded stge fter 48 hours culture. Cryoprotectnt --Dy_7 Age of Dy--8 embryo Dy9- ANOVA EME 87.3(71) 55.3(38) 43.5(23) E G 76.5 (68) 80.6 (36) 39.5 (43) DEG 68.3 (60) 57.1 (21) 20.8 (24) P G (43) 70.0 (40) 35.0 (20) ANOVA b Vlues in rows nd columns with no letters in common re significntly different ~<0.01). EME~Ethylene glycol monomethyl ether; EG~Ethylene glycol; DEG~Diethylene glycol; PG~Propylene glycol. Figures in prenthesis indicte number of embryos. Tble 3 shows the survivl rtes bsed on the bility to htch from the zon pellucid fter 48 h of culture. As to the ge of embryo, the htching blstocysts rtes in the Dy-9 embryos were significntly lower thn those in the Dy- 7 nd 8 embryos mong 4 cryoprotectnts (P<0.05). As to the cryoprotectnts, there were significnt differences between DEG nd EME, between DEG nd EG nd between PG nd EG (P<0.05).

5 Theriogenology 919 Tble 3. Survivl rtes (%} of in vitro produced bovine blstocysts frozen with vrious cryoprotectnts. Survivl rtes bsed on the bility to htch from the zon fter 48 hours of culture. Cryoprotectn t -,~-~---=Accg~e_o~f,--'e-=mccb~r""y_o =-_ Dy 7 Dy 8 Dy 9 AN OVA EME 66.2(71) 44.7(38) 34.8 (23) E G DEG P G 60.3 (68} (60} 51.2(43) (36} 33.3(21) 40.0 (40) 34.9 (43) (24) 20.0 (20) ANOVA b Vlues in rows with no letters in common re significntly different (P<0.05). -b, c-d Vlues in columns re significntly different (P<0.05). EME=Ethylene glycol monomethyl ether; EG=Ethylene glycol; DEG=Diethylene glycol; PG=Propylene glycol. Figure in prenthesis indicte number of embryos. DISCUSSION Mssip et l. (9} nd Suzuki et l. (15) hve lredy succeeded in freezing in vivo bovine embryos by direct trnsfer method. Since then, mny reserchers hve pplied these methods for in vivo or in vitro fertilized embryos. Recently, Voelkel et l. (17, 18} used ethylene glycol to freeze in vivo embryos nd obtined high survivl nd pregnncy rtes by direct trnsfer. In the present experiment, bovine IVF embryos on Dys 7, 8 nd 9 were frozen in PG, EG, DEG nd EME. The methods of direct rehydrtion into holding medium without step-wise or sucrosemedited dilution of ech cryoprotectnt were exmined to determine the effects of different embryo ges. Tchikw et l. (16} reported tht the verge percentge of htching embryos ws decresed with the dely of development to the blstocyst stge. Fukushim et l. (4} reported tht the pregnncy rte of IVF bovine embryos on Dy 8 ws lower thn on Dy 7. Similrly, in our study the verge survivl rte nd the percentges of expnding nd htching blstocysts were decresed with the progression of dys in 3 of 4 cryoprotectnts (excluding EG}. The present findings suggest tht the survivl rte of the frozen-thwed bovine embryos is ffected by the embryo qulity nd not the type of the cryoprotectnt used. Voelkel et l. ( 17, 18} reported tht poor in vivo ( 16%} nd in vitro (30.2%} embryo vibility ws observed when PG ws used s cryoprotectnt. However, in our experiment, the verge rte of reppernce of blstocoele (81.4%}, nd the percentges of fully expnded (65. 1 %} nd htching (51. 2%} blstocysts in the frozenthwed IVF bovine embryos on Dy 7, using PG s cryoprotectnt, were not significntly different from those obtined using EG (85.3%, 76.5% nd 60.3%, respectively} s cryoprotectnt. The difference observed in the vibility of frozen-thwed bovine embryos my be due to the different freezing methods used (with different seeding point, cooling rte etc.). The IVF embryos used in this experiment were co-cultured with cumulus cells ccording,c b d

6 920 Theriogenology to the methods of Goto et l. (5). Fukui et l. (3) reported tht the cell number of the blstocysts would be effected depending on the in vitro culture condition. This suggests tht the differences in survivl rtes between our experiment nd tht of Voelkel et l. (17,18) my be dependent on the differences in culture conditions, embryo qulity, embryo ge nd whether they re fertilized in vivo or in vitro. When IVF embryos on Dys 7 nd 9 were frozen using EME s the cryoprotectnt, the verge rte of reppernce of blstocoele nd the percentges of expnding nd htching blstocysts were higher thn those obtined using the other cryoprotectnts. These findings indicte tht EME my be useful cryoprotectnt for bovine embryos, with direct rehydrtion performed by plcing thwed embryos into holding medium. However, more reserch is needed to determine possible negtive effects of EME. In conclusion, the present findings suggested tht embryo ge (Dy 7 nd 8) is very importnt fctor for successful freezing of IVF bovine embryos. As regrds htching rte of frozen thwed bovine IVF embryos, fter in vitro culture, EME nd EG my be superior s cryoprotectnts thn PG nd DEG. REFERENCES 1. Bilton RJ, Moore NW. Successful trnsport of frozen cttle embryos from New Zelnd to Austrli. J Reprod Fertil 1977;50: Brckett BG, Oliphnt G. Cpcittion of rbbit spermtozo in vitro. Biol Reprod 1975;12: Fukui Y, Ono H. In vitro development to blstocyst of in vitro mtured nd fertilized bovine oocytes. Vet Rec 1988;122: Fukushim M, Toming K, Hty Y, Utsumi K. Vibility of frozen-thwed bovine blstocyst stge embryos mtured fertilized nd developed in vitro. Jpn J Anim Reprod Technol. 1991;14: Goto K, Kjihr Y, Kosk S, Kob M, Nknishi Y, Ogw K. Pregnncies fter co-culture of cumulus cells with bovine embryos derived from in vitro fertiliztion of in vitro mtured folliculr oocytes. J Reprod Fert 1988;83: Kjihr Y, Goto K, Kosk S, Nknishi Y, Ogw K. In vitro fertiliztion of bovine folliculr oocytes nd their development up to htched blstocysts in vitro. Jpn J Anim Reprod 1987;33: Kuwym M, Hmno S. Cryopreservtion of bovine blstocysts developed from in vitro fertilized oocytes mtured in vitro. Jpn J Anim Reprod Technol 1991;13: Leibo SP. A one-step in situ dilution method for frozen-thwed bovine embryos. Theriogenology 1984;21:

7 Theriogenology Mssip A, Vn Der Zwlmen P, Ectors F. Recent progress in cryopreservtion of cttle embryos. Theriogenology 1987;27: Mtsuok K, Skt S, Ichino K, Shimy Y, Suzuki T. Effect of superovulted cow serum for culture of bovine oocytes to the blstocyst stge. Theriogenology 1992;37:254 bstr. 11. Mukoujim S, Skguti T, Otni T, Yoneym H. In vitro fertiliztion of bovine oocytes ins eminted with spermtozo treted by different methods nd trnsfer ppliction to field. Jpn J Anim Reprod Technol 1990;12: Niemnn H, Scher B, Schilling E, Smidt D. Improvement of survivl rtes of bovine blstocysts with sucrose for glycerol dilution fter fst freezing nd thwing method. Theriogenology 1982;17:102 bstr. 13. Renrd JP, Heymn Y, Leymonie P, Plt JC. Sucrose dilution: technique for field trnsfer of bovine embryos frozen in the strw. Theriogenology 1983;19:145 bstr. 14. Suzuki T, Shimohir I, Fujiym M. Trnsfer of bovine frozen embryos by one step strw method.,jpn J Anim Reprod 1983; 29: Suzuki T, Ymmoto M, Ooe M, Skt A, Mtsuok M, Nishikt Y, Okmto K. Effect of sucrose concentrtion used for onestep dilution upon in vitro nd in vivo survivl of bovine embryos refrigerted in glycerol nd 1,2-propnediol. Theriogenology 1990;34: Tchikw S, Otoi T, Kondo S. One-step dilution of frozen -thwed bovine embryos derived from in vitro fertilized oocytes using propylene glycol s cryoprotectnt. Jpn J Anim Reprod Technol 1991;13: Voelkel SA, Hu YX. Direct trnsfer or fro~an-~h~wad bovine embryos. Theriogenology 1992;37: Voelkel SA, Hu YX. Use of ethylene qlycol s cryoprotectnt for bovine embryos llowing direct trnsfer of frozen-~hwed embryos to recipient femles. Theriogenology 1992;37: Whittinghm DG. Survivl of mouse embryos fter freezing nd thwing. Nture 1971;233: Wilmut I, Rowson LEA. The successful low temperture preservtion of mouse nd cow embryos. J Reprod Fertil 1973; 33: