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|Title:||EFFECT OF KISSPEPTIN ON IN VITRO MATURATION OF BUFFALO OOCYTES|
|Abstract:||This study was aimed at investigating the effect of kisspeptin (Kp) on In Vitro maturation of buffalo oocytes. The oocytes were aspirated from the ovaries collected from the nearby slaughter house. From a total of 1371 ovaries, 2400 culture grade oocytes were collected with a mean of 1.78 oocytes per ovary. Two different experiments were conducted to investigate the effect of Kp alone at different concentrations (experiment 1) or in combination with other hormones viz... Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Estradiol (E2) and IVM media (experiment 2) on In Vitro maturation of buffalo oocytes. In the first experiment, oocytes were allowed to mature in media supplemented with three different concentrations (5, 10 and 15 μg/ml) of Kp, with the sole purpose of arriving at an appropriate concentration of Kp to be used in experiment-2. Tissue Culture Medium 199 (TCM 199B), supplemented with Gentamicin was used as control. Culture grade oocytes xxi were randomly allotted to different In Vitro maturation media and cultured at 39oC with 5% CO2 under humidified atmosphere for 24 hours. At the end, oocytes were evaluated for Cumulus Cell Expansion (CCE), extrusion of 1st Polar Body (PB). The proportion of oocytes showing CCE and extrusion of 1st PB was highest in the medium supplemented with 10 μg/ml of Kp. Subsequently, experiment-2 was designed to evaluate the effect of Kp (10 μg/ml) on In Vitro maturation of buffalo oocytes, in combination with various hormones. In this experiment, oocytes were allowed to mature in 12 different maturation media viz. T1 to T12 [Control (TCM-199); T1, Kp; T2, FSH; T3, FSH+Kp; T4, LH; T5, LH+Kp; T6, E2; T7, E2+Kp; T8, FSH+LH+E2; T9, FSH+LH+E2+Kp; T10, IVM; T11, IVM+Kp; T12]. Kp, FSH, LH were supplemented at the concentration of 10 μg/ml each while E2 was used at 1 μg/ml concentration in experiment-2. IVM medium consists of TCM-199 was supplemented with FSH (10 μg/ml), LH (10 μg/ml), E2 (1μg/ml), Fetal Calf Serum (10%) and Bovine Serum Albumin (10 μg/ml). The proportion of CCE and extrusion of PB has also significantly increased when Kp was supplemented with FSH (86.05±0.44 and 63.99±0.48), LH (81.34±0.54 and 59.94±0.46) and E2 (63.98±0.48 and 53.33±0.80). The maturation rates increased significantly further when FSH, LH and E2 combination (T9) were additionally supplemented with Kp (T10). However, they were significantly lower than T11 (IVM) and as well T12 (IVM+Kp). To conclude, the present study demonstrated that In Vitro addition of Kp (10 μg/ml) to FSH, LH and Estradiol supplemented media enhances buffalo oocyte maturation In Vitro.|
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