Successful transport of frozen cattle embryos from New Zealand to Australia.

Several laboratories have reported the birthofyoung from frozen-thawed mouse embryos transferred to recipient females (Whittingham, Leibo & Mazur, 1972; Wilmut, 1972) and frozen mouse embryos have been successfully transported from North America to the United Kingdom (Whittingham & Whitten, 1974). Of the domestic species, continued development in vivo and in vitro has been achieved with cattle (Wilmut & Rowson, 1973; Bilton & Moore, 1976a), sheep (Moore & Bilton, 1976; Willadsen, Polge, Rowson, & Moor, 1976) and goat (Bilton & Moore, 1976b) embryos stored in liquid N2, and the potential value of frozen storage for the transport of embryos has been noted. During March 1976, cattle embryos were collected at Christchurch, New Zealand, from mature parous cows ofmixed dairy breeds 7-8 days after A.I. with semen from a Simmental bull. All embryos were morulae or early blastocysts and after collection they were frozen in Dulbecco's phosphate buffer enriched with 25% bovine blood serum (DB+S) and containing 1-5 M-dimethylsulphoxide (DMSO) or 1-0 M-glycerol. DMSO was added at 30°C and the embryos were moved through a series of dishes containing increasing concentrations of DMSO (0-25,0-5, 0-75,10,1-25, 1-5m) in DB+S, remaining in each dish for 4-5 min. Glycerol, when used, was added at 37°C by a more direct procedure. DB+S containing 2-0 M-glycerol was added over a period of 20 min to the embryos held in an equal volume of DB+S devoid of glycerol. After the addition of DMSO or glycerol, the embryos together with 2-3 ml of their respective medium were transferred to Pyrex glass freezing tubes (75 10 mm) and cooled to 0°C at a rate of 0-7°C/min. The tubes were then cooled to —50°C at rates of 013 or 0-3°C/min and crystallization was initiated at —3°C by the addition of crystals of frozen DB+S. Once the tubes had reached —50°C they were cooled more rapidly (l°C/min) to —105°C and then transferred to liquid N2. The embryos were then transported in liquid N2 containers by air to Sydney, Australia, and then by road (120 km) to Mittagong. After storage in liquid N2 for 2-3 months the embryos were thawed as described by Bilton & Moore (1976a) at one of three rates (Table 1) measured over the range -50°C to 0°C. The tubes were then warmed to 30°C (0-7oC/min) and the DMSO and glycerol were removed by dilution. DMSO was removed by the reverse of the procedures used for its addition, whilst glycerol was diluted by the slow addition over 15 min of three volumes of DB+S. After dilution the embryos were washed twice in fresh DB+S and then cultured in DB+S at 37-5°C for 12 h (Bilton & Moore, 1976a). The embryos were thawed in two batches. In the first, 11 embryos stored in DMSO were thawed but none showed any development in culture. It was subsequently found that the serum used in the dilution procedures and culture medium was at fault: DB+S prepared from this serum failed to