Effect of addition and removal rates of cryoprotectant on the survivability of frozen bovine embryos

THERIOGENOLOGY

M. Taneja,S.M.Totey,GurpreetSingh,C.H. Pawsheand G.P. Talwar J&nbryo Biotechnology Laboratory, NationalInstitute of Dmmology, JNU Canplex,ShahidJeet Singhm, New Delhi110 067 INDIA Thisexperiment was desi9nedto studythe effectof rate of glycerol addition (5-stepor 3-step)priorto the freezing of bovine eabryosand rateof its subsequent removal(5-step or 2step with or withoutthe additionof sucrose in the dilution msdim) on theirpost-thawswzvival (PTS). Fortyfiveembryoscollectednon-surgicallyonday 7 (day0= onsetof estrus)fran 9 supemvulatedHolstein-Friesian cows kiere used for this study.EhWyos of similardevelopmntalstage (late mrulae to expanded blastocysts)and embryo quality grade (kexcellentand2=good)werera&anly assignedtooneof the four treatments. In Trt A,B ahd C the embryosware exposedto glycerol (1.4M) in PBS (with10% fetalcalf serum)by a 5-step addition (0.25M, 0.5OM,0.75M,l.OOMand then 1.4OM),whilein Trt D by a 3-step addition (0.5OM, l.OOMand then 1.4OM),with a 10 min equilibration (25'C)at eachconcentration in all the treatmsnts. Wryos pipettedintostrawswBre thenplacedintoa programnsble freezer unit (Bio&ol",FTS Systm, NY). The freezing sequence was: 25°C to -7°Cat l"C/min; seeding; 0.5'C/minto -3O'C; 0.3"C/mihto -38°C;plungihgintoliquidnitrcgen.EWzyos were thawed in a 37°Cwaterbath.In Trt A and B glycerolwas removed by a 5-stepdilutionin a reverseorderof its addition, with a 10 min equilibration at each step.The only difference in Trt B was the presenceof l.OMsucroseat eachcohcehtration. In Trt C and D embrycs ku3refirstpipettedintoamixtureof 0.7M sucrose and 0.7M glycerol (total concentration 1.4M)for 7 min and theh transferred intoa 0.7Msucrosesolutionfor 2 min. Dilutions mre follomd by several washesi&PBS inall the treatments.Wryos wareassessedforPTSonthebasisofnmpholo9y and waregraded (l=excellent to 4=degenerate). Resultsare sumnarized below: Treatment A

n (%)

TreatnmntB Treatmentc TreatmehtD n ($1 n 0) n (%)

Excellent 0.00 3/11 (27.3) 2/10 (20.0) 4112 (33.3) 6/12 (50.0) 3/11 (27.3) 5/10 (50.0) 4112 (33.3) E&rate

3/12 (25.0) 0.00 5/11 (45.5) l/10 2/10 (10.0) (20.0) 2112 2/12 (16.7)

The xona damageratewas 41.7,45.5,30.0 and 16.7% for Groups A,B,Cand D respectively. Datawere analyzed by Fisher's exact test.Therewere ho significant differences either in the overall PTS rates or in the zona damage rates between the treatmentgroups, Hoover the rapid a-step dilution method appeared to providehigherpercentageofgradeland 2 ss&ryos with lawerzonapellucida damagerates. 30%

JANUARY

1992 VOL. 37 NO. 1