The Production of Elite Breezing Stock by Embryo Multiplication

Summary

The aim ofthe project was to produce large numbers oflow cost, genetically identical, elite cattle embryos for transfer into recipient cows to generate genetically identical, elite offspring. The embryos were to be produced by novel, simplified nuclear transfer procedures using embryonic cells as the source ofdonor nuclei.

Considerable progress was made towards meeting the project goals.

Using simplifiednuclear transfer and cryopreservation techniques developed during the project, it was demonstrated that over 70 transferable (morula and blastocyst stage), recycled nuclear transfer embryos could be generated from one original embryo during three rounds ofrecycling.

Nine cloned calves derived from embryonic cells were born during the project including two sets ofgenetically identical bull calves. There are a further 25 diagnosed cloned pregnancies generated late inthe project awaiting calving data.

The production ofnuclear transfer embryos and pregnancies using somatic cells such as fetal fibroblasts as the source of donor nuclei was added as an objective because ofrapid advances being made inthis area overseas.

Eighty-seven somatic cell nucleartransfer morula and blastocysts for transfer were generated inthe last sixmonths ofthe projectusing microinjection techniques. These embryos were transferred, either fresh or after cryopreservation, into recipient cows, and generatedthree pregnancies.

Vitrificationtechniques for the cryopreservation ofearly stage nucleartransfer embryos, developed and successfully applied during the project, have the potential to overcome the logistical problems associated with multiple rounds ofnuclear transfer. Thesenovel vitrification methods will also allowthe cryopreservation and direct transfer ofin vitro produced

embryos produced by cloning, whichis a prerequisite for widespread adoption ofthe technology.

The absence ofabnormalities, or grossly oversized calves inthe cloned offspring born so far inthe project is an advance on overseas experience. However, the birth ofmore calves will be requiredbefore it canbe assumed that the nuclear transfer and culture techniques developed during this project do not leadto abnormal offspring.

Approximately 50% ofnucleartransfer pregnancies positively diagnosed byultrasound around 35 days ofgestation were lo~t later in gestation. Losses ofthis magnitude are not observedinin vitro produced embryos. Whether the problems causing suchlosses are placental or fetal in origin, itis likely that they stem from inadequate reprogramming ofthe donor nucleus following its transfer into a recipient cytoplast.

A considerable amount ofscientific data inrelationto cloning has beengenerated during this project. This includes information onrecycling, donor and recipient cell cycle stages, cryopreservation and reprogramming ofboth embryonic and somatic cells innuclear transfer.

The Project sawthe successful transfer oftechnology from Monash University to Genetics Australia. This has included establishing an efficient productionprocess at Genetics Australia for the maturation ofbovine oocytes andfor the production ofinvitro embryos.

Systems were also developed for the management ofrecipients prior to and after embryo transfer and for managing calving and calves after delivery, which maximize outcomes from embryo transfer.

Umavellingthe mechanisms involved innuclear reprogramming may hold the key to the production ofnuclear transfer embryos which result in ongoing pregnancy rates acceptable for widespread agricultural applications ofthe technology.

There are, however, applications ofthe technology inwhich lower pregnancy rates will give commercially viable outcomes. These applications are high value added products such as genetically enhanced animals, elite beefbulls, or elite dairy cows.

Project MU035 whichfollows MU02I aims to answer ftmdamental scientific questions about nuclear reprogramming and its effect onembryo viability andpregnancyrates. Itwill evaluate outcomes from present efficiencies with embryo recycling combined with vitrificationtechniques, and will continue to develop the efficiencies possible. Theproject will continue to develop and evaluate somatic cell nuclear transfer as a source ofdonor cells, including testing embryo viability and pregnancy results following recycling embryos generated from somatic cells. Itwill evaluate outcomes possible by combiningthese techniques withtransgenic enhancement.