BOVINE EGG TRANSPLANTATION - PRESERVATION OF EMBRYOS

  • 1 January 1980
    • journal article
    • research article
    • Vol. 32  (12) , 523-532
Abstract
The development of methods for deep freezing blastocyst stages of mice, sheep and cattle is briefly reviewed. Dimethylsulfoxide (DMSO) and glycerol have so far been the most widely used cryoprotectants for embryo freezing. In the initial studies, DMSO was mostly used but the latest results seem to indicate that glycerol might be more successful. The pH variation and osmotic changes induced by glycerol seem to create less stress on the embryo from an analytic point of view. A biological freezer especially designed for embryo freezing was used in all trials. The following 5 different freezing trials are described. With slow freezing (0.3.degree.-0.1.degree. C/min) down to -60.degree. C, slow thawing at 4.degree. C/min, and cryoprotectant (1.5 M DMSO), 41 embryos were frozen; 30 (73%) survived after thawing; 19 were transferred surgically to heat synchronized recipients and 9 (42%) became fertilized. The 2nd trial involved slow freezing (0.3.degree.-0.1.degree. C/min) down to -40.degree. C, slow thawing at 8.degree. C/min or quick thawing in water at 37.degree. C and cryoprotectant, 1.5 M DMSO; 45 embryos were frozen; 65% survived slow thawing and 23% survived quick thawing. Glass ampoules and French straws gave similar survival rates. A combination of 0.5 M DMSO and 1.0 M glycerol was used in the 3rd trial combined with slow freezing (0.3.degree.-0.1.degree. C/min) down to -40.degree. C. Slow thawing (8.degree. C/min) was applied; 29 embryos were frozen and 12 (41%) survived slow thawing. The 4th trial encompassed 1.0 M glycerol as the cryoprotectant, slow freezing (0.3.degree.-0.1.degree. C/min) down to -30.degree. C and -33.degree. C, and direct thawing with H2O at 37.degree. C. Nineteen embryos were frozen, 50% and 44% survived in the 2 groups. The 5th trial involved freezing (0.3.degree. C/min down to) at -30.degree. C, -35.degree. C or -40.degree. C, with cryoprotectant 1 M glycerol; number of embryos/survivors in the 3 groups was 9/6 (67%), 12/10 (83%) and 5/3, (60%), respectively. Seven of the embryos frozen to -35.degree. were transferred cervically to recipients and 5 (71%) became fertilized. No pregnancies were established when 4 freshly collected blastocysts were transferred to recipients in 0.6 M glycerol/phosphate buffered saline. In the control group without 0.6 M glycerol, 2 of 4 recipients became pregnant. The temperature for addition and removal of glycerol (20.degree. C or 37 C) had no effect on the survival rate. Apparently the feasibility of storing cattle embryos in liquid N is well established and the import and export of cattle blastocysts will increase in the future.