الفهرس 
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Abstract
Vitrification is a common method for cryopreservation of gametes and embryos. Although successful oocyte vitrification has been achieved in several animal species, subsequent progress is still limited especially in buffalo. This limitation is due to the alterations which may take place during vitrification process. The goal of this study is to establish an efficacious vitrification protocol which can overcome the alterations in buffalo oocytes during vitrification. This study include some experiments for evaluation of meiotic chromosome in vitrified matured buffalo oocytes, effect of combination of cryoprotectants such as DMSO, EG at two concentration 6M and 7M, cryodevices such as straw and Cryotop and addition of antioxidant as cysteamine to maturation media on the out come of vitrification process.
Evaluation of survivability and meiotic competence in vitrified matured buffalo oocytes: the in vitro matured oocytes were vitrified in a mixture of EG+ DMSO, then the meiotic stages were evaluated after warming and staining by orcein stain. Manipulation of oocytes and vitrification procedures were performed at room temperature (25oC). The vitrification solution (V1) was 1.5 M EG + 1.5 M DMSO, the vitrification solution 2 (V2) was 3 M EG + 3 M DMSO. Cryoprotectants were added in 2 steps, using half the final concentration (in the second step) for the first step. The state of nuclear maturation was determined, oocytes that reached telophase I or metaphase II stages were considered matured. Post thaw maturation rate (matured oocyte ⁄ survived oocyte) was calculated. The morphologically normal, viability and the percentage of matured oocytes were significantly higher in control than matured vitrified oocytes.
Effect of cysteamine during in vitro maturation on viability and meiotic competence of vitrified buffalo oocytes: the experiment evaluate the effect of addition of cysteamine as antioxidant to maturation media on oocytes and vitrification outcomes. The vitrification solution was the same as experiment 1. Vitrified oocytes matured in cysteamine free and 50 M cysteamine were compared. Each treatment was vitrified in 0.25-ml straws. The maturation rates were significantly higher in cysteamine group than without cysteamine. The percentage of oocytes reaching MII in the control group was significantly higher than of those vitrified after maturation in culture media with or without cysteamine.
Effect of using cryodevices on vitrification of in vitro matured buffalo oocytes: Concerning the effect of cryodevices on viability and maturation of vitrified matured buffalo oocytes, the in vitro matured oocytes were divided into two groups, the first one was vitrified using conventional French straw, while the other was vitrified using Cryotop. The vitrification solution was the same as experiment 1. Maturation rates of vitrified thawed buffalo oocytes were significantly higher in Cryotop than straw. The survival rate after vitrification was similar for both straw and Cryotop. The percentages of viable oocytes were significantly lower in straw and Cryotop than in control.
Effect of two concentrations of cryoprotectants on vitrified buffalo oocytes: To evaluate the effect of two concentrations of cryoprotectants on vitrification of in vitro matured buffalo oocytes, a mixture of DMSO and EG as CPAs solutions were prepared in TCM-199 medium with two concentrations as a final concentration . The first concentration was 3M:
1.5M E.G +1.5M DMSO and 6M: 3M E.G + 3M DMSO. The second concentration was 3.5 M:1.75 M DMSO + 1.75M EG and 7M: 3.5 M DMSO + 3.5 M EG. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. The survival rate after vitrification was similar for both concentrations (6M and 7M) groups. The maturation rates of vitrified thawed buffalo oocytes were significantly higher in concentration of 7M group than media with 6M.