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Monitoring and assessment of nuclear transfer pregnancies using maternal pregnancy recognition proteins.
thesisposted on 14.02.2017, 01:10 by Utama, Suzanita
Abnormal fetal and placental development is common in somatic cell nuclear transfer (SCNT). Abortion rates and perinatal mortality of cloned calves are high. Early identification of abnormal pregnancies followed by early termination would reduce the risks to recipients and costs of maintaining cloned pregnancies unlikely to result in a normal calf. Protein biomarkers in the blood were studied seeking a predictor of abnormal fetal and/or placental development. Bovine Placental Lactogen (bPL), activin A, follistatin and bovine Pregnancy Specific Protein B (bPSPB) in peripheral maternal blood were targeted as marker candidates. Monthly concentrations of these proteins and changes in concentrations over time were examined in cows pregnant with cloned embryos. bPL profiles showed that if detectable amounts of bPL are not present by month 3, it is unlikely that the cow has a viable fetus. Detectable amounts of bPL at month 3 may indicate that pregnancy might be maintained at least up to month 8. At month 4, the tendency to a high bPL concentration may indicate impending abortion at month 8. Activin A tended to rise just before abortion. A relatively high activin A to follistatin ratio at month 1 and month 2 may indicate pregnancies destined to reach tenn. PSPB concentration varied considerably at all stages of SCNT and IVF pregnancies. PSPB tended to decline in cloned pregnancies one month before abortion. Interferon-tau (lFN-r) was investigated by means of Western Blot of the induced Mx protein from the circulating leukocytes. Technical problems encountered indicated the need for a larger amounts of sample. A more specific and pure primary antibody is needed. Quantitative RT-PCR may be able to overcome the problems. Novel pregnancy specific proteins that might be used as markers were investigated by comparing protein maps of pregnant and non pregnant plasma. The high dynamic range of blood proteome interfered with the screening. Together with individual animal variation, it is nearly impossible to perform global blood comparative proteomic with the current techniques. Global blood protein expressions of full term cloned and IVF pregnancies were compared using Two-Dimensional fluorescence differential gel electrophoresis to simultaneously find some protein marker candidates. Comparison between cloned pregnancy that reached term and cloned pregnancy that was aborted showed as many differentially expressed proteins as those between cloned and IVF full term pregnancies. No differences were found in blood proteins between cloned pregnancies that went to term and cloned pregnancies that were aborted. This could suggest that these term calves were not in fact normal at the genome level as others have found and as was indicated by a high perinatal mortality. No single biomarker of abnormal pregnancies was found. Further work should concentrate on refinement of the cloning techniques and the type of abnormalities studied. Combining assessment of some or all of the blood constituents studied here may lead to a multiple marker for impaired pregnancies.