Parthenogenetic activation is a technology to initate the artificial stimulation of oocyte development without fertilization and it is a vital method to study biochemical mechanisms in the fertilization process, intracytoplasmic sperm injection (ISCI) technology, and nuclear transfer technology. The aim of the presented study is to stimulate the developmental process of non-fertilized oocyte artificially and to apply spectroscopic methods for examining the activation stimulated with different ways.
Non-activated oocytes were recovered from unmated female mice after superovulation with human chorionic gonadotropin (hCG) injection. For chemical activation, the oocytes were separately exposed in 5% solution of Ethanol for 7 minutes, and in 0.05mM Strontium Chloride solution (SrCl2) (Sigma-Aldrich, USA) for 45 minutes. “Young” oocytes of 15hrs post hCG injection and “aged” oocytes of 21hrs post hCG were subjected to activation with SrCl2 in order to confirm the best age condition of oocytes to do artificial activation. Chemically-treated oocytes were compared with non-activated control, which also can reveal spontaneous parthenogenetic activation.
The efficiency of different chemical agents for the parthenogenetic activation and further development has been estimated via routine microscopic/morphological method of the oocyte conditions evaluation and it was estimated on reaching following stages: forming of polar body (PB), polar body extrusion, and 2-cell stage. From our result, spontaneous activation had lower efficiency compared to chemical activation because the development of parthenogenotes was not triggered by any activation agents.
The results have been compared with Raman spectroscopic analysis of the non-activated oocytes and activated oocytes using 532 nm and 785 nm laser wavelength excitation, varying laser power and time exposure. Raman spectroscopic study of the oocytes on different stages of activation included measurements of spectra in different morphological areas of the oocytes, analysis of characteristic peaks variations and spatial distribution of their intensity along the oocyte in dependence on stage for oocytes activated at various experimental conditions such as chemical agents, timing, and age of oocyte.
The pronounced Raman bands in the 900-3400 cm-1 region are observed for most living cells, but the intensity and exact spectral position vary for different morphological areas of the oocytes; especially in Amide I range (1640-1680 cm-1) and range including Amide III peak, some lipids vibrations (1245-1325 cm-1) gives complimentary information for the oocyte analyzing. Re-distribution of intensity of peaks of Cytochrome C (1130 cm-1, 1314 cm-1 and 1585 cm-1) along oocyte is observed during activation and characterizes the energy metabolism during activation and development processes.
Using excitations in visible and near IR ranges allows to select the experimental conditions of the highest safety and for the acquisition of the most informative spectra. Analysis of the spectra allows to select the criteria for estimation of the oocyte conditions and to discuss establishment of new method for the investigation of early mammalian development.

Keywords: Raman Spectroscopic; Parthenogenetic; Oocyte activation

Acknowledgements i
Abstract ii
Content iv
Index of figure v
Tables vii
Chapter 1 Introduction 1
1.1. Assisted reproductive techonology 1
1.2. Parthenogenetic development 3
1.3. Raman Spectroscopy 5
1.4. Research Motivation 6

Chapter 2 Methodology 7
2.1. Collection of oocytes 7
2.2. Experimental group 8
2.3. Raman measurements 8
Chapter 3 Result and Discussion 9
3.1. Age-related in oocytes activation 9
3.2. Oocytes artificial activation 11
3.3. Raman spectroscopy investigation 13
Chapter 4 Conclusion 32

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