الفهرس 
INTRRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Although buffaloes are one of the main sources of meat and milk in Egypt and some developing countries, these animals suffer from low productive and reproductive potentials due to many problems such as Late sexual maturity, silent heat, seasonal anoestrus and long periods of postpartum ovarian inactivity resulting in extended calving intervals which cause great economic losses in milk and/or meat production.
FSH one of the pituitary gonadotropins which have an integral role in the regulation of reproductive activity in buffalo. Due to the importance of FSH to the reproductive performance of the animals, many attempts for isolation and purification of FSH from different animals were done. However, scanty studies and literatures were published concerning buffalo gonadotropins extraction and purification. Therefore, the present study aimed for isolation and purification of buFSH from buffalo pituitary glands, detection of its purity and biological potency and studying its pharmacological effect on buffalo oocytes maturation in vitro, for further use in buffalo in vivo to improve its reproductive performance.
Buffalo pituitary glands were collected from GERCO abattoir in Al basattin, transported with ice bags to the laboratory and stored at -20 oC.
Two phases were applied for isolation of buFSH from buffalo pituitary glands. The first phase, precipitation of pituitary gonadotropins by ammonium sulphate with change molarity and pH. The final product of this phase was crude buFSH. The second stationary phase was purification by gel filteration chromatography using sephadex G-100 and the protein concentration of the resulted fractions was estimated and finally three peaks were obtained.
For verification of the purity of the isolated buffalo pituitary extract, the lyophilized fraction containing buFSH was applied to (SDS-PAGE), stained with Commassie Brilliant Blue (CBB), a single band was obtained which assure purity.
The purified buffalo pituitary buFSH was used in superovulation of immature mice with different doses compared to different doses of PMSG to determine its biological potency in albino mice. Standard curve of PMSG was drawn to detect the activity of the purified buFSH.
Purified buffalo pituitary buFSH was added to maturation media of buffalo oocytes during IVM with different doses, the results showed that there was a significant difference compared with the control. The addition of purified buFSH improved the maturation rate of buffalo oocytes in vitro.
17β-estradiol (E2) was added to maturation media of buffalo oocytes during IVM plus purified buFSH, the results showed that there was no significant difference compared with using of purified buFSH alone.
β-mercaptoethanol (β-ME) was added to maturation media of buffalo oocytes during IVM plus purified buffalo pituitary buFSH, the results showed that there was a significant difference compared with using of purified buffalo pituitary buFSH alone. The addition of β-ME improved the maturation rate of buffalo oocytes in vitro.
It can be concluded that:-
Buffalo pituitary follicle stimulating hormone (buFSH) can be isolated from buffalo pituitary glands with high yield in pure form based on ammonium sulphate precipitation and purification on column chromatography.
Use of pure form of buFSH represent a significant improvement over previously used methods of superovulation, enabling large numbers of mice oocytes to be obtained that are capable of normal fertilization and development during the preimplantation stages.
Additon of purified buFSH to the maturation medium containing buffalo oocytes improve the maturation rate of buffalo oocytes in vitro.
Addition of E2 plus purified buFSH did not cause any additional improvement in maturation rate of buffalo oocytes.
Addition of β-ME to maturation media of buffalo oocytes containing buffalo purified buFSH increased the maturation rate of buffalo oocytes compared to maturation media of buffalo oocytes containing purified buFSH alone.
Taking into consideration, it can be suggested that purified buFSH may be used to improve the reproductive efficiency of buffaloes in vivo and production of healthy oocytes capable for maturation and fertilization by natural manner.
It is needed to separate the gene coding FSH for biotechnology production and make trials for sequencing of buffalo FSH gene and production of recombinant buffalo FSH used for improving fertility of buffaloes especially for superovulation and in vitro fertilization.