99 Increased expression of microRNAs in sperm of Nelore Bulls with high in vitro fertility

Abstract

The spermatozoon is no longer known only as a cell that delivers the male genetic material to the oocyte, because it also provides molecules such as microRNAs (miRNAs) that play a significant role in fertilization and embryonic development. MiRNAs are small noncoding RNAs capable of modulating mRNA translation, thus affecting important biological processes. Sperm miRNAs may influence embryo development and therefore, might be related to in vitro production of embryos (IVP), considering that individual bulls have different fertility rates when used for IVF. The aim of this work was to identify miRNAs expressed in semen of bulls with high and low IVP rates. The composition of groups was based on a retrospective database from a reproductive biotechnology company between the years of 2016 and 2018, generating around 7000 IVP manipulations of 430 Nelore bulls. We only considered IVP manipulations that used a minimum of 30 oocytes and conventional semen selection by Percoll gradient. A total of 87 Nelore bulls fit these criteria. We then ranked bulls based on cleavage rate (number of cleaved structures/number of oocytes), blastocyst rate (number of blastocysts/number of oocytes) and embryo development rate (number of blastocysts/number of cleaved structures). Considering these three rates, we allocated bulls to two groups. The top eight were considered to have high IVP fertility (HF) and the bottom eight were grouped together as low IVP fertility (LF). We performed the T TEST procedure (SAS 9.3 software; SAS Institute Inc.) to compare the groups for cleavage (P < 0.0001), blastocyst (P = 0.0006), and embryo development (P = 0.0001) rates. For miRNA analysis, sperm were separated using Percoll gradient and subjected to RNA extraction and cDNA synthesis. First, quantitative real-time PCR was used to evaluate the abundance of 380 bovine-specific miRNAs in a pool of samples for each group, using QuantStudio 6Flex. Then, 48 miRNAs presenting at least a 3-fold change of normalized cycle threshold values between groups were selected: 23 highly detected in HF, 1 highly detected in LF, 18 exclusively detected in HF, and 2 exclusively detected in LF. Last, we evaluated the abundance of these selected miRNAs in each experimental unit. We identified four miRNAs highly abundant in sperm from HF bulls, bta-miR-10a, bta-miR-383, bta-miR-93, and bta-miR-449b. Our results suggest that these miRNAs could play important roles in bovine embryo development.

This work was supported by FAPESP (grant# 2018/03871-6).