Assessment of Total Oocyte Transcripts Representation through Single Ooplasm Biopsy in Bovine with High Reliability

Abstract

Understanding the entire transcriptional and epigenetic landscape is facilitated by the application of omics in a number of ways. Today, omic instruments are more affordable and easier to implement. In human research, for instance, single-omics are a reality and are used extensively to generate vast quantities of data. This method permits the comprehensive reconstruction of transcriptome and epigenetic markers removing bias from pooled samples. In tandem with the evolution of machines and protocols, algorithms and genome annotation have undergone continuous improvement. The genome annotation of domestic animals is inferior to that of humans, rodents, and less complex organisms. In the case of heifers, the reference is incomplete, with significant gaps and only a portion of the noncoding transcripts. The purpose of this study is to validate our compartmentalized single oocyte biopsy by comparing a small fraction of bovine oocytes, 1%, to the entire oocyte at the Metaphase II stage. In addition, we examined the use of four database sources (NCBI, ENSEMBL, UCSC, and NONCODE) to produce a merged non-redundant gene alignment and counting in order to enhance gene detection and normalization, resulting in a more accurate method to comprehend the entire landscape. This study is a continuation of our research titled "Retrospective model utilizing biopsies, granulosa cells, and polar body to predict oocyte competence in bovine" in which this method was used to retrospectively compare biopsy oocytes collected during the MII phase. With the addition of NONCODE information, gene normalization was significantly enhanced. In addition, our analysis identified 4560 noncoding genes from NONCODE references. ENSEMBL and NCBI have nearly the same number of annotated genes (16,423 vs. 17,804), but using ENSEMBL as a reference, 2356 genes were able to be normalized and identified. Proceeding to biopsy x oocyte analysis, we were able to detect a greater number of genes in oocytes than in biopsy, where the preponderance was from NONCODE sources (68). Despite these minor differences, the high correlation of expression between them (89%) was consistent and proved to be a valuable instrument for studying the oocyte without destroying it.