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Authors: Yadav, Jitender
Advisor: Nanda, Trilok
Title: Expression profile of Cyclin B gene in buffalo cumulus oocyte complex
Publisher: LUVAS
Language: en
Type: Thesis
Keywords: Cyclin B, IVM, COCs, Real time PCR, Cloning, Sequencing
Abstract: The present study was carried out to determine the temporal expression of Cyclin B gene in follicles of different size using real time PCR. From 1257 ovaries, 740 and 1881 good quality COCs were recovered by aspiration from large (>6 mm) and medium (2-6 mm) follicle respectively. The recovered COCs were matured in TCM-199 medium supplemented with 10% FBS, 0.4% BSA and hormones FSH and LH, along with controls without FBS and BSA, and incubation at zero min, 30 min, 60 min, 90 min, 18 hr, 20 hr, 22 hr, 24 hr and 26 hr in a CO2 incubator with 5% CO2 under humid conditions at 38.5°C. The maturation was assessed by the expansion of cumulus cells of COCs. From in vitro matured COCs total RNA was isolated, reverse transcribed and amplified by Cyclin B gene specific primers. Real time PCR was carried out to study the expression pattern of Cyclin B gene in follicles of different sizes. The 318 bp amplified product was purified and cloned successfully into pGET cloning vector using E. coli cell JM107 strain as the host cell. Positive cloneswere sequenced and the phylogenetic relationship of Cyclin B gene of buffalo was determined with other species. Result: The recovery rate of COCs per ovary was 2.08. The overall IVM rate of COCs was 81.90% and 50.21% in TCM-199 supplemented with FBS and BSA and control. The expression of Cyclin B was higher in medium follicles. The expression of Cyclin Bwas highest at zero min in both large and medium follicles and decreases as the maturation time increases.In the phylogenetic analysis, the Cyclin B geneof Bubalus bubalis are found more closely related to that of Bos taurusthan to Rattus norvegicus. Conclusion: Function of Cyclin B is more during early phases of maturation and less during further stages of maturation.
Subject: Animal Biotechnology
Theme: Expression profile of Cyclin B gene in buffalo cumulus oocyte complex
These Type: M.V.Sc.
Issue Date: 2012
Appears in Collections:Thesis

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