AVALIAÇÃO DA EXPRESSÃO DE microRNAs ESPERMÁTICOS ASSOCIADO A TAXAS DE PRENHEZ E PERDA GESTACIONAL EM IATF

Abstract

The growing demand for animal protein underscores the importance of early identification of bulls with high genetic potential. Such identification promotes reproductive and productive improvement of herds and enables the global commercialization of semen with high economic value traits. Reproductive molecular markers have high commercial value as they allow early diagnosis and validation of genetic potential. Their use drives the international market for animal genetics and artificial insemination. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression. Spermatozoa-derived miRNAs can help identify causes of subfertility or idiopathic infertility through qPCR analysis. These miRNAs regulate gene expression and are directly linked to reproductive quality. Due to this role, they are considered promising fertility biomarkers and may be useful for reproductive diagnosis in both animals and humans. This study aimed to evaluate changes in the expression of miRNAs associated with pregnancy per insemination (P/AI) rates and gestational loss in bulls used in fixed-time artificial insemination (FTAI), based on field data. A total of 29 bulls were selected from a database according to their field fertility performance, grouped by the highest and lowest 10% pregnancy and gestational loss rates. Total RNA was extracted from cryopreserved semen doses following a protocol registered with the INPI. RNA concentration and quality were assessed via spectrophotometry. From the extracted total RNA, cDNA was synthesized using the TaqMan™ Advanced miRNA cDNA Synthesis Kit, and qPCR assays were performed with TaqMan™ probes specific for miR-7, -204, -139, and -191. Samples were analyzed in duplicate to obtain the mean Ct for each sample. The 2^-ΔΔCT method was applied to assess changes in miRNA expression, with statistical differences between groups evaluated by t-test for individual groups and one-way ANOVA followed by Tukey’s post hoc test for combined groups, with significance set at p < 0.05. miR-7 showed increased expression in low P/AI samples compared to high P/AI, without influence from gestational loss, whereas miR-204 showed a trend (p = 0.08) toward higher expression in high gestational loss samples, becoming significant in low P/AI samples (p < 0.05). miR-191 differed in expression between two commercially relevant groups: high fertility (high P/AI and low gestational loss) and low fertility (low P/AI and high gestational loss). Male fertility-associated miRNAs are being investigated, as in this study, for their potential as predictors of male reproductive capacity, which holds great commercial interest for the development of biotechnologies.