130 Expression of selected biomarker candidate genes to confer in vitro maturation in Indian buffaloes

Abstract

In vitro maturation (IVM) of oocytes is a crucial step and is directly related to better embryo production in buffaloes. Therefore, we planned to study gene expression of GDF9, HAS2, SPRY1, ARHGAP22, COL18A1, and GPC4 genes in IVM and immature cumulus–oocyte complexes (COCs). The COCs were recovered from follicles of slaughter origin ovaries of native buffaloes. COCs were observed under stereo zoom microscope and categorized in four grades according to morphology. Of the four grades, the first three grade COCs were considered and randomly allotted in two groups: immature treatment group (n = 263) and IVM treatment group (n = 272). IVM of COCs was carried out in 100-μL drops of BO-IVM medium overlaying embryo tested oil in a 35-mm petri dish under 5% CO₂ in a 39.0°C incubator for 24 h. Cumulus of COCs of both groups were removed by treating with 0.25% trypsin, and oocytes were stored in RNALater for future use. The expression of genes was evaluated using quantitative PCR, and the relative expression of each gene was calculated using the ΔΔ^Ct method with efficiency correction. The logarithmic transformation of fold change (log2FC) of each candidate gene in the IVM oocyte group was computed against the immature oocyte group based on the observed cycle threshold values. Appropriate standard deviations were determined based on the observed deviations among the triplicates. The expression in the IVM treatment group of previously reported upregulated genes (GDF9, HAS2, SPRY1) was higher (up to 10-fold) compared with the immature treatment group (reference group). In the present study, relatively lower expression was observed for the other candidate genes (ARHGAP22, COL18A1, GPC4) in the bovine transcripts of oocyte, which were previously also reported as being downregulated.