Principles and practice of freezing bovine embryos

Abstract

This dissertation reviews the principles and practice of mammalian embryo preservation with particular reference to bovine embryos. The sections include principles of freezing, sensitivity of some embryos cooling, early methods of freezing mammalian embryos, four modern methods currently used, integration of various methods into practice and assay of survival.

A study has been carried cut in an attempt to simplify the procedure of freezing and thawing. Embryos were recovered on day 7-6 from cross-bred heifers which had bean induced to super ovulate by injection with PMSC. Some heifers also received an experimental injection of anti serum to PMSC. All the fertilized embryos which were recovered were frozen, regardless of their morphology.

The embryos were allocated to four different treatment groups. In experiment I embryos were cooled in three different ways after being cooled at 0.3°C min. from -7°C to -33°C. They were either plunged directly into liquid nitrogen, held for 30 minutes, or cooled to -36°C at 0.1°C min. prior to plunging. Thawing was by agitating the test tubes containing the embryos in a water at 37° C until the ice disappeared,

Experiment 2 was a pilot trial to confirm whether embryos could tolerate direct transfer into and out of a medium containing 10% glycerol and either 0.25 or 0.5 M sucrose. In experiment 3, embryos were frozen and thawed in the presence of 10 per cent glycerol and either 0.25 or 0.5 Ml sucrose. After seeding at -7°C, embryos were cooled at 0.2°C to -15, -20, -25 or -30°0. After thawing, glycerol was removed by direct transfer of embryos into 1 ml of PBS.

In Experiment 4, embryos were cooled in the presence of 10% glycerol and either 0.25 or 0.5-1*1 sucrose followed by direct plunging into liquid nitrogen. Glycerol was removed either by direct transfer of embryos into PBS or by transfer into a medium containing sucrose at the same concentration followed by transfer into PBS.

The survival rate of embryos cooled using three methods (Exp.l) was poor. Direct plunging at -33°C gave the same survival rate as plunging at -36°C after being cooled at 0.1 C min⁻¹. from -33° C. By contrast none of the embryos survived when held for 30 minutes at -33° C before plunging into liquid nitrogen.

The result of the pilot trial confirmed that embryos can survive when transferred into and out of a medium containing 10% glycerol and either 0.25 or 0.5 M sucrose (85% survival). However, experiment 3 and 4 revealed that cooling embryos either at 0.2° C min. to -15, -20, -25 and -30° C or at 0.3°C mini to -33 followed by plunging into liquid nitrogen is not compatible with survival irrespective of the way the glycerol was added and diluted.

It was concluded that holding embryos before plunging them into liquid nitrogen can be damaging. It was also suggested that the quality of embryos before freezing is critical for their future development after freezing and thawing.

Although embryos frozen in the presence of sucrose did not survive, it was suggested that the cooling and warming procedures should be varied in search of a satisfactory method