Cryoprotectants and gene expression of cryopreserved oocytes and embryos in buffalo / Esraa Aly Sayed Ismail ; Supervised Mohamed A. I. Elsayed , Nabil A. Hemeida , Omaima M. Kandil

Thesis (Ph.D.) - Cairo University - Faculty of Veterinary Medicine - Department of Theriogenology

This work was carried out for four successive years (2018, 2019 , 2020 and 2021) in order to investigate the effect of different cryoprotectant agents on 1) vitrified/thawed buffalo oocytes developmental competence, 2) viability of vitrified/thawed transferable buffalo embryos and 3) gene expression of in vitro vitrified/thawed matured buffalo oocytes and embryos. Excellent and good oocytes were cultured in TCM-199 medium at 38.5 {u00BA}C, 5% CO2 for 22 hrs, then fertilized using frozen semen and cultured on SOF media. Matured oocytes or transferable embryos were vitrified in DMSO, EG or combination of DMSO+EG by two steps procedure and stored in LN. The morphologically normal vitrified/thawed in vitro matured oocytes were fertilized using frozen semen and cultured on SOF media for detection the cleavage and transferable embryos rates to assess its viability. Fresh matured buffalo oocytes/embryos and recovered vitrified/thawed buffalo oocytes/ embryos were stained with Hochest stain to assess the stage of maturation of in vitro matured oocytes or cell count of in vitro produced embryos and stained with Mitotricker red for detection the mitochondrial distribution and intensity. mRNA was extracted from vitrified/thawed buffalo in vitro matured oocytes or in vitro produced embryos vs fresh matured oocytes or embryos using Picopure kits, cDNA was synthesized by QuantiTect Reverse Transcription kits

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