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Development of a system to cryopreserve bovine embryos produced in vitro in a simple culture medium

In vitro produced embryos are not marketed frozen due to
special conditions that render them unable to survive after
freezing. Technology used for embryos in co-cultures will be
improved and applied to a simple, semi-defined medium.

By using conventional MOET technology, a donor embryo yields 5 viable embryos every 2 months on average, whilst the in vitro production (IVP) system combined to repeated ovum pick up (OPU) makes it possible to obtain 20 viable in vitro produced embryos (IVP) embryos within the same time interval. However, most often, unlike in vivo embryos, which are commercialized frozen and exchanged worldwide, IVP embryos cannot be transferred in this way due to low survival rates after transfer of frozen embryos into recipients. This feature avoids optimal marketing, since it shortens the lifespan to transfer the embryo to a recipient (24 hours approximately for fresh embryos), and renders the product perishable. In addition, a higher number of synchronised recipients are necessary, which increases costs. Finally, cryopreservation makes it possible to carry out all the sanitary investigations currently in use for in vivo produced embryos and thus guarantees a safe transfer in reference to identified diseases. Even when satisfactory, co-culture as an embryo production system is imperfect as compared to culture in defined or semi-defined conditions Synthetic Oviduct Fluid (SOF) system. The SOF system is safer and repeatable, promotes a higher reduction of sanitary risks (e.g. BSE and viruses), and maintenance is cheaper and more friendly than a growing cell co-culture. In addition, calves born from SOF-produced embryos have lower chance of experiencing Large Offspring Syndrome (LOS). Applied research will be done through this project to better understand the detrimental effects of in vitro production on the freezing ability of the embryos. This research will lead to develop more simple tools to measure such effects and to develop culture systems based on defined media making it possible to freeze IVP embryos and achieve good pregnancy rates after transfer. The French partner (UNCEIA) has developed a freezing procedure based on a co-culture system which recently permitted a 50% pregnancy rate after transfer of 63 IVP frozen embryos emanating from slaughterhouse ovaries. The licence for this procedure has now been submitted for acceptance (file 0007096). The project will involve the following tasks: Task 1) Implementation of the Vero-cells freezing system as a starting point in SPAIN: The most frequent scientist and technical exchange will take place over this period. IVP embryos will be produced in SPAIN and FRANCE. Sets of embryo transfers in SPAIN will concern embryos produced and frozen both locally and from SELIA (local control). At the same time, a set of embryos produced and frozen by SELIA will be transferred in FRANCE (external control). Task 2) Modifying culture conditions for freezing: Additives and changes in culture conditions applied to the SOF system will lead to embryos with a diminished lipid stock in embryonal cells, which is to a higher extent responsible for the reduced cryopreservation ability of IVP bovine embryos. Both reduction of lipid synthesis and increased mobilisation approaches will be rounded off in FRANCE and SPAIN and primarily tested by analysing lipid contents and in vitro embryo survival after cryopreservation. Task 3) Intramural production and transfer of frozen embryos: Embryos will be produced, frozen and transferred under an applicative context to the experimental herd. Frozen embryos derived from conventional MOET procedures will be used as a control group. Careful monitoring of pregnancies and the checking for normalcy and health of calves born will determine subsequent on field trials. Task 4) On farm embryo transfer of frozen embryos: An embryo transfer trial with IVP and MOET embryos frozen in the laboratory will be conducted over dairy farms from potential users of the developed technology. Pregnancy, calves status and acceptance by farmers will be controlled using a specialised recording datanet. Keywords: bovine in vitro, embryo freezing.
Acronym: 
EMFREEZE
Project ID: 
2 573
Start date: 
01-06-2001
Project Duration: 
61months
Project costs: 
2 260 000.00€
Technological Area: 
Animal Selection/Production / Husbandry technology
Market Area: 

Raising the productivity and competitiveness of European businesses through technology. Boosting national economies on the international market, and strengthening the basis for sustainable prosperity and employment.