الفهرس 
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Abstract
The present study was carried out at the Laboratory of Physiology and biotechnology belonging to the Animal Production Department, Faculty of Agriculture, Minufia University in cooperation with The International Livestock Management Training Centre (ILMTC) belong to Animal Production Research Institute (APRI), Agriculture Research Centre, Ministry of Agriculture during the period from spring 2011 to August 2013.
Three experiments were conducted in order to study the following:-
1- Effect of supplementation of antioxidants (100, 150 and 200μM GSH) and (100, 200 and 300μM α-tocopherol) to maturation media on cumulus cells expansion and nuclear maturation rates of buffalo oocytes (Experiment 1).
2- Effect of supplementation of antioxidant (1, 2 and 3 mM GSH) and (1, 2 and 3 mM VE ) to diluted fresh semen as well as the duration of semen storage at 5°C for five days on spermatozoa motility, spermatozoa livability and normality as well as hypo-osmatic swelling test (HOST) of buffalo semen (Experiment 2).
3- Effect of the best level of antioxidants supplementation of GSH and VE either to the maturation media or to semen extender as results of experiments 1 and 2 suggested on the fertilization rate and embryo development stages to the blastocyst stage (Experiment 3).
Experiment: 1
Effect of antioxidants supplementation to the maturation media on cumulus cells expansion and nuclear maturation rates of buffalo oocytes:
Buffaloes ovaries were collected from Shebin El-Kom slaughterhouse, the previous history of the reproductive performance of slaughtered animals were unknown. The collected ovaries were placed immediately after slaughtering into thermos in saline solution (0.9% NaCl) supplemented with antibiotics (100 IU penicillin and 100μg streptomycin/ml) at 30°С. The collected ovaries were transported to the laboratory within 0.5-2h.
In the laboratory, the excess tissues were cut from the ovarian stalk and the ovaries were washed three times with warmed phosphate buffer solution (PBS) supplemented with antibiotics (100 IU penicillin and 100μg streptomycin/ml) to remove clotted blood. Then all ovaries were washed quickly one time with ethanol (70%) to remove any contamination on the ovarian surface.
Visible follicles on the ovarian surface with 2-8 mm in diameter were counted. Oocytes were collected by aspiration technique, using a 18-gauge hypodermic needle attached with a sterile disposable 5 ml syringe containing 1 ml harvesting medium (PBS). After aspiration, contents of the syringe were placed into Petri dish (30 x 60 mm) for searching oocytes under stereomicroscop.
After collection, oocytes were washed three times in harvesting medium, thereafter oocytes were counted and evaluated under inverted microscope and classified according to their cumulus morphology and ooplasm homogeneity and into four grades as follow:
Grade (A): characterized by dark homogenous ooplasm and more than four compact layers of cumulus cells.
Grade (B): characterized by dark homogenous cytoplasm and more than four layers of less compact cumulus cells starting to expand peripherally.
Grade (C): characterized by heterogeneous granulated ooplasm.
Grade (D): oocytes completely or partially denuded of cumulus cells.