الفهرس 
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Abstract
This work was carried out at Physiology laboratory, Animal Reproduction Research Institute (ARRI), Agricultural Research Center, Al-Haram, Giza, Egypt, in cooperation with Animal Production Department, Faculty of Agriculture, Ain Shams University, Egypt.
During the period from November 2012 to January 2015 six experiments were conducted to test and establish a standard method for cryopreservation of bovine oocytes using vitrification.
Generally, ovaries were collected directly after slaughtering from local abattoir. There after the oocytes were transported to laboratory in thermo flask containing normal physiological saline. Oocytes were aspirated from follicle (2-8 cm in diameter). Oocytes were washed three times in TCM199 then examined under stereo microscope for oocytes selection. Morphologically normal oocytes with an evenly granulated cytoplasm and a compact cumulus cell layer were selected.
Morphologically normal oocytes equilibrated in equilibration solution (which is half concentration of vitrification one). After a period of equilibration, oocytes were transported to vitrification solution. Oocytes of all experiments were maintained in the vitrification solution (VS) with different concentrations and combinations of cryoprotectants for few seconds. During this time (few seconds), oocytes were loaded into different cryodevices (straw, open-pulled straws, or solid surface vitrification). And different concentrations of L-Carnitine were adding to maturation medium in experiment 6. Then cryodevices plunged into liquid nitrogen (LN2). After a period of storage, oocytes were warmed and subjected to assessment of quality using different methods (assessment of morphology, staining for viability, fertilization rate, and developmental competence).
The obtained results could be summarized as follow.
Experiment 1: Effects of different concentrations of Propanediol (PROH) in the equilibration solution on morphology, viability and fertilization rate of vitrified-warmed mature bovine oocytes
The effect of different concentrations of propanediol (2.5%, 5%, and 10%) on morphology, viability, fertilization rate on vitrified-warmed mature bovine oocytes. This experiment demonstrated that 5% PROH resulted in the best quality vitrified-warmed oocytes as indicated by higher percent (65.85%) normal morphology, (92.59%) viability rate and (40.0%) fertilization rate. While, 10% of group resulted (35.89%) normal morphology, (64.28%) survival rate and (22.22%) fertilization rate, also 2.5% PROH resulted 55.56%, 86.67% and 30.77%, in the corresponding values respectively.
Experiments 2: Effects of different concentrations of DMSO in equilibration solution on morphology, viability and fertilization rate of vitrified-warmed mature bovine oocytes
The effect of different concentrations of DMSO (10%, 20%, and 40%) on morphology, viability, fertilization rate in vitrified-warmed mature bovine oocytes was studied. 20% DMSO revealed significantly high survival and fertilization rates (90.63% and 44.83%, respectively), While 10% and 40% DMSO were resulted in lower percentages.
Experiment 3: Effects of different concentrations of EG in equilibration solution on morphology, viability and fertilization rate of vitrified-mature bovine oocytes
The effect of different concentrations of EG (10%, 20%, and 40%) on morphology, viability, fertilization rate in vitrified-warmed mature bovine oocytes was studied. 20% EG revealed significantly high survival and fertilization rates (91.54%, and 44.61%, respectively), While 10% and 40% EG were resulted in lower percentages.
Experiment 4: Effects of different combinations of cryoprotectants on viability, fertilization rate and embryo development rate of vitrified-warmed mature bovine oocytes
The effect of different combinations of cryoprotectants (20%EG + 20%DMSO), (20%EG + 5%PROH), (5% PROH + 20%DMSO) and (5%PROH + 20%DMSO + 20%EG) on morphology, viability, fertilization rates and developmental competence were studied. This experiment recorded higher percent (65.85%) normal morphology, (92.59%) survival rate and (40.0%) fertilization rate. While, 10% group resulted in (35.89%) normal morphology, (64.28%) survival rate and (22.22%) fertilization rate, also 2.5% PROH resulted in 55.56%, 86.67% and 30.77%, respectively.
(20%EG + 20%DMSO) resulted in significantly higher on the development rate to the blastocyst stage (92.45%, and 57.14%, respectively) than other groups.
Experiment 5: Vitrification of matured bovine oocytes using different cryodevices (OPS, 0.25 ml plastic straws, and SSV)
The effect of different cryodevices using combination of 20%EG and 20%DMSO was studied. In this experiment OPS and SSV techniques reported higher viability (94.73%, and 95.23%), fertilization rate (61.11%, and 67.50%), and cleavage rate (24.32, and 31.25%) than 0.25ml semen straw techniques.
Experiment 6: Effect of different concentrations of L-carnitine in maturation medium on morphology, viability, fertilization rate and embryo development of vitrified-warmed mature bovine oocytes
The effect of different concentration of L-Carnitine (control,0.1,0.3,0.5) on morphology, viability, fertilization rate and embryo development was studied. In this experiment, 0.3mmol concentration of L-Carnitine resulted in significantly higher normal morphology, viability and fertilization rate (70.18% - 92.11% and 62.86%), respectively than control and the other concentrations. Also embryo development cleavage, morula, blastocyst rate (40.48% -19.05% - 11.90%) were higher than other concentrations.
CONCLUSION
1. 5% PROH is better than both 10% and 20% PROH for vitrification of mature bovine oocytes.
2. 20% EG is better than both 10% and 40% EG for vitrification of mature bovine oocytes.
3. 20% DMSO is better than both 10% and 40% DMSO for vitrification of mature bovine oocytes.
4. 20% EG + 20% DMSO is the best combined mixture for vitrification of mature bovine oocytes.
5. Use of SSV and OPS is better than 0.25 ml semen straw for vitrification of mature bovine oocytes.
6. 0.3 mM/ml L-carnitine concentration with 20% EG + 20% DMSO loaded into SSV is better than control, 0.1 and 0.5 for vitrification of mature bovine oocytes.
The current study indicated that the combination of 20% EG and 20% DMSO showed greater effectiveness in the vitrification of mature bovine oocytes. The viability of vit¬rified bovine oocytes could be improved us¬ing SSV as cryodevice compared with OPS and Straw methods. An appropriate level of L.C (0.3 mM/ml) in oocytes maturation medium can induce molecular changes associated with oocytes maturation, leading to increased oocyte developmental potential and improved blastocyst quality. Further research would be needed to clarify the effect of L.carnitine on bovine oocytes.