الفهرس 
المقدمةيوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
Hyaluronic acid (HA), is an important component of the extracellular matrix and plays a crucial role in cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor (CD44) are expressed in cumulus oocyte complexes (COCs) in different animal species and increased during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. 4methylumbelliferone (4-MU) inhibits synthesis of hyaluronic acid.
The main objective of this study is to investigate the effects of the molecular size and concentration of HA on cumulus cells expansion, in vitro nuclear maturation and development competence of buffalo oocytes. Studying the effect of addition of HA (0.1, 0.5 mg/mL), Hyal2 (10, 100, 300 U/mL), 4-MU (0.1, 0.5, 1 mM), or a combination of HA 0.5 mg/mL + Hyal2 300 U/mL and HA 0.5 mg/mL +4-MU 1 mM in maturation medium on cumulus cell expansion and in vitro nuclear maturation of buffalo oocytes.
Also studying further embryo development after in vitro fertilization by studying the effect of addition of (HA 0.5 mg/ mL, Hyal2 300 U/mL, 4-MU 1 mM, HA 0.5 mg/mL + Hyal2 300 U/mL, HA 0.5 mg/mL +4-MU 1 mM) in maturation medium of buffalo oocytes..
Oocytes were collected from ovaries of slaughtered animals. Compacted oocytes were matured in vitro in (TCM-199 medium supplemented with different treatments) for 22 h in CO2 incubator at 38.5 °C, 5% CO2 and 95% humidity. Maturation rate was determined, in terms of degree of expansion of cumulus cells and oocytes at metaphase-II (nuclear maturation).
In fertilization experiments, compacted oocytes were matured in vitro in TCM-199 medium supplemented with different treatments for 22 h in CO2 incubator at 38.5 °C, 5% CO2 and 95% humidity. After in vitro maturation, the cumulus oocyte complexes (COCs) were gently pipetted to remove adhering granulosa cells and to break up aggregated COCs. After that, the oocytes were washed in the fertilization medium then transferred to the fertilization medium contained two million sperm cells/ ml) for 18 h in CO2 incubator at 38.5 °C, 5% CO2 and 95% humidity.
After in vitro fertilization, presumptive zygotes were gently pipetted to remove adhering granulosa cells. After that, zygotes were cultured in synthetic oviductal fluid medium that contain amino acids, citric acid and myo-inositol with adding bovine serum albumin (0.4% w/v) and then placed for 7 days in CO2 incubator at 38.5 °C, 5% CO2 and 95% humidity. Medium was changed each two days. Cleavage and blastocyst rates were recorded.
Results of this study indicate that the addition of hyaluronic acid in maturation medium did not affect either rate of the cumulus expansion or development competence of buffalo oocytes (cleaved and blastocyst stage) but, it affected in oocyte nuclear maturation rate.
Results also showed that the addition of Hyal2 alone or in presence of exogenous hyaluronic acid in maturation medium resulted insignificant inhibition of cumulus expansion compared with control group, but it leads to increase in oocyte nuclear maturation rate compared with control group. Also did not significantly affect the rate of cleaved and blastocyst stage.
The results also showed that inhibition of hyaluronic acid synthesis with different concentration of 4methylumbelliferone (4-MU) is significantly hinder cumulus expansion, and also leads to decrease oocyte nuclear maturation rate and further embryo development which totally leads to blastocyst completely inhibition compared with each treatment, an effect which was partially abrogated by exogenous HA supplementation.
CONCLUSION
We conclude from those results that the production of hyaluronic acid by cumulus cell during maturation is an important matter not only for cumulus cell expansion, but also for oocyte nuclear maturation and developmental competence of buffalo oocytes. This effect is independent on molecular size of hyaluronic acid, and there is no relationship between both cumulus cell expansion and in vitro buffalo oocyte nuclear maturation.