الفهرس 
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Abstract
The present study aimed to improve invitro fertilization of buffalo oocytes by testing the effect of supplementation of IVF and IVC media with different concentrations of Spirulina platensis extraction, EGCG, myo-inositol, divalent chelators and the effect of different sperm- oocyte incubation time (4-6h., 14-16h, 21-24h.).
Buffalo ovaries were collected from a local abattoir then transported to the laboratory within 2 h. Good quality cumulus oocytes complexes (COCs) were aspirated from medium sized follicles using an 18-gauge needle. selected COCs were washed three times in sterile phosphate-buffered saline (PBS) then cultured for 24 h in tissue culture medium (TCM)-199 supplemented with 10% fetal bovine serum (FBS), 0.005 IU/mL follicle stimulating hormone (FSH) , and 1 mg/mL 17β-estradiol at 38º c in a humidified atmosphere of 5% CO2. Separation and selection of motile sperm were done using swim-up technique. Matured oocytes were coincubated with sperm (106 spermatozoa / mL) for 18 h in Tyrode’s albumin lactate pyruvate (TALP)-IVF medium. The presumptive zygotes were cultured in synthetic oviductal fluid (SOF) medium at 38º c in a humidified atmosphere of 5% CO2. Developmental competence of embryos was assessed separately for each group every 48h and the culture medium were replaced with fresh medium. Evaluation the early buffalo embryo was performed using MTT assay and total cell number.
Experiment I. Effect of Spirulina platensis extraction on in vitro fertilization and the quality of developed embryo: Generally the supplementation of fertilization and culture media with Spirulina platensis extraction improve both fertilization rate and embryo development specially 100μl/ml and 50 μl/ml.
Experiment II .Effect of epigallocatechin-3-gallate: Incorporation of epigallocatechin-3-gallate in IVF technique in concentration of 5mg/ml markedly improved fertilization rate and embryo quality and this represented by its effect on total cell number and on mitochondrial function of embryo.
Experiment III. Effect of myo-inositol: Enrichment of IVF and IVC media with 5, and 10mg/ml increase fertilization rates and embryo quality.
Experiment IV: Effect of divalent cations: Combination of 10 µl/ml of stock solution (containing different concentrations of L.histine, L-cysteine, and d-penicillamine) with fertilization media (TALP) noticeably improves penetration and fertilization rates and embryo quality.
Experiment V: Effect of sperm- oocyte incubation time: 21-24h sperm-oocyte incubation is ideal for optimizing fertilization rates of buffalo oocytes.
Conclusion: The efficient recognition of the molecular contents of the oviduct that augment fertilization in vivo may help in efficient in vitro culture systems that improve embryo viability.