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THE DEVELOPMENT REFINEMENT AND COMMERCIALISATION BASED ON THE IN-VITRO PRODUCTION, SEX DETERMINATION, FREEZE STORAGE AND TRANSFER OF BOVINE EMBROYONS

Objective

The objective of the proposed research is to achieve industrial scale production of sex determined bovine embryos of high genetic merit, which are pathogen free and can be stored frozen for nonsurgical transfer. Embryo transfer in cattle is currently based on the use of superovulation and uterine flushing to recover embryos. The yield is low and variable resulting in a high embryo cost which inhibits the widespread commercialization of current techniques.
The aim of this project was to develop new, and to refine existing biotechnology, for the industry scale production of in vitro fertilized bovine embryos of specific genotype. These would be cultured in vitro to the blastocyst stage, be sex determined and frozen stored for transfer to recipient cows on an international scale.
An in vitro embryo production system has been established with consistent rates of maturation, fertilization and embryo development to the blastocyst stage. The yield of transferable embryos per ovary have been increased. A 1-step freezing/thawing system has been developed with a consistent hatching rate (over 80%) of Day 7, grade 1-2 embryos after 72 hours of postthaw culture. High pregnancy rates and twinning rates have been obtained from fresh in vitro fertilization (IVF) embryos after transfer to nonbred recipients or bred recipients. A high pregnancy rate has also been achieved with frozen thawed embryos after transfer, but the twinning rate is still low.

The aim of this project was to develop new, and to refine existing biotechnology, for the industry scale production of in vitro fertilized bovine embryos of specific genotype. These would be cultured in vitro to the blastocyst stage, be sex determined and frozen stored for transfer to recipient cows on an international scale.
A successful in vitro culture system for cattle ova to support development from 1-cell to blastocysts was developed. This was based on coculture with oviduct cells. Physical contact between ova and cell monolayers is not essential for high rates of ovum development. The immunosurgical method developed highlighted the fact that in vitro cultured embryos have similar total inner cell mass and trophoblast cell number to embryos recovered in vivo at the same stage.

The aim of this project was to develop new, and to refine existing biotechnology, for the industry scale production of in vitro fertilized bovine embryos of specific genotype. These would be cultured in vitro to the blastocyst stage, be sex determined and frozen stored for transfer to recipient cows on an international scale.
The polymerase chain reaction (PCR) has revolutionized many area of molecular genetic analysis over the past couple of years. We have used this process in the development of new genetic markers and in the detection of residual malignant disease. In the latter area, probes derived from human chromosome Y are in routine use which are capable of generating male specific signals from no more than a few cells. Conditions have been optimized for the amplification of a bovine autosomal sequence using PCR. The use of fluorophores for the labelling of amplification products are under study. Processes for bisection and/or biopsy of embryos using micromanipulation have been successfully refined.
The project research tasks are as follows:

Develop ovarian storage conditions to allow flexibility in the timing of oocyte recovery; increase oocyte recovery numbers by developing ovarian culture to increase follicle numbers; enzymatic digestion to remove all oocytes.

Undertake studies to maximize the oocyte maturation and fertilization rates. Such studies will include sperm capacitation, storage of capacitated sperm and novel in vitro fertilization methods.

Develop an in vitro embryo culture system to support growth from single cell to blastocyst stage and a rapid routine method for sex determination of bovine embryos.

Undertake studies on the freeze storage of in vitro fertilized embryos.

Optimize pregnancy and twinning rates using in vitro fertilized embryos by studying the effect of embryo distribution and by applying different approaches to twin pregnancy diagnosis.

Assess the biological and economic efficiency of twinning in dairy cows.

The Community dairy herd not only produces milk but also 80% of the calves used in the beef industry. This herd is contracting following the imposition of milk quotas resulting in fewer calves being available for beef production. Twin calf production would reduce this calf deficit. The proposed in vitro biotechnology will significantly reduce embryo costs, making twin induction by embryo transfer a cost effective approach. This in turn will have a real impact on the supply of calves for beef. The weight of calf weaned per beef cow with twins is increased by 60% compared with a beef cow rearing one calf, requiring an extra energy input of only 12 to 15%. The transfer of specialized beef embryos to that portion of the dairy herd not required for replacement breeding will increase the supply of suitable beef calves.

A further and significant improvement will result from the transfer to these cows of sexed, male embryos. The ability to move cattle as frozen embryos makes it more feasible to capitalize on the animal genetic diversity which exists worldwide. A second important advantage emerging from this biotechnology is the safety of in vitro washing as a method of ensuring both virus and pathogen free embryos.

Coordinator

Ovamass Ltd
Address
County Tipperary
45 Fethard
Ireland

Participants (3)

Estação Zootécnica Nacional
Portugal
Address
Fonte Boa
2000 Vale De Santarém
TEAGASC - AGRICULTURE AND FOOD DEVELOPMENT AUTHORITY
Ireland
Address
19,Sandymount Avenue 19, Ballsbridge
4 Dublin
THE PROVOST, FELLOWS AND SCHOLARS OF THE COLLEGE OF THE HOLY AND UNDIVIDED TRINITY OF QUEEN ELIZABETH NEAR DUBLIN HEREINAFTER TRINITY COLLEGE DUBLIN
Ireland
Address
Smurfit Institute,trinity College
2 Dublin