STUDY OF KISSPEPTIN EFFECT ON IN VITRO MATURATION OF SHEEP OOCYTES
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Date
2016-10-25
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PVNR TVU
Abstract
The aim of this study was to investigate the effect of kisspeptin (Kp) on in vitro maturation of sheep oocytes. Sheep oocytes were aspirated from ovaries collected at slaughter house. From a total of 1226 ovaries, 1920 culture grade oocytes were collected with a mean of 1.56 oocytes per ovary. Two different experiments were conducted to investigate the effect of Kp alone (experiment 1) or in combination with Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Estradiol (E2) (experiment 2) on in vitro maturation of sheep oocytes. Tissue culture medium 199(TCM 199B) supplemented with Gentamicin was used as control medium. Culture grade oocytes were randomly allocated to different in vitro maturation media and cultured at 39oC in 5% CO2 under humidified atmosphere for 24 h. At the end of in vitro maturation, the oocytes were evaluated for their Cumulus Cell Expansion (CCE) and extrusion of 1st Polar Body (PB) and presence of MII stage chromosomes. In experiment-1, oocytes were matured in three different concentrations (5, 10 and 15
21
μg/ml) of Kp supplemented media. The proportion of oocytes showing CCE, extrusion of PB was highest, when the oocytes were matured in the medium supplemented with 10 μg/ml of Kp.
Based on the best concentration resulting from experiment-1, experiment 2 have been conducted to evaluate the effect of Kp with different combinations of the hormones on in vitro maturation of sheep oocytes. In experiment-2, oocytes were matured in twelve different maturation media (T1-T12: Control: 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 (10 μg/ml), FSH (10 μg/ml), LH (10 μg/ml) and E2 (1 μg/ml) were supplemented wherever required in experiment-2. IVM medium was supplemented with FSH (10 μg/ml), LH (10 μg/ml), E2 (1μg/ml), Fetal calf serum (10%) and Bovine serum albumin (BSA) (10 μg/ml).
In experiment-1, the effect of different concentrations of Kp (5, 10 and 15 μg/ml) on in vitro maturation of sheep oocytes was investigated. The proportion of oocytes showing CCE and extrusion of PB as indicator of MII stage chromosomes were highest in medium supplemented with Kp (10 μg/ml).
In experiment-2, the proportion of oocytes showing CCE and extrusion of PB was highest (98.33±1.05 and 89.17±2.38), when they were matured in IVM+Kp (T12) and it was statistically higher than all other treatments (T1-T11). The proportion of CCE and extrusion of PB was significantly increased when Kp was supplemented to FSH (94.17±0.83 and 69.17±3.27) and E2 (67.50±1.71 and 59.17±2.39) but no effect was observed in the presence of LH (89.17±3.00 and 64.17±3.01). The maturation rates were significantly increased when FSH, LH and E2 (T9) was additionally supplemented with Kp (T10), but it was significantly lower than IVM+Kp (T12). In conclusion, the present study demonstrated that the addition of Kp (10 μg/ml) to the FSH, LH and Estradiol supplemented medium enhance the sheep oocyte maturation in vitro.
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extraction, diseases, planting, plant extracts, pathogens, bacteria, sowing, fungi, biological phenomena, irrigation