Differential expression of selected microRNAs in cryopreserved sperm of Bos taurus sires with diverse fertility status

Abstract

Our study aimed to evaluate the expression of four selected microRNAs (miRNA) in commercially produced cryopreserved semen of bulls with high vs. low fertility. The annual fertility index (FI) of 151 sires with ≥300 first services was recorded between November 2018 and November 2019. Sires lying at the extremes of the FI distribution (outside the range of mean FI±SD) were classified as low- (LF; n=13 bulls) or high-fertile (HF; n=7 bulls), with a non-return rate of 61.4±6.9% and 72.6±1.5%, respectively. For each bull, total RNA was isolated using a modified TRIzolTM-based protocol from two cryopreserved semen batches produced with 1-mo interval (40 sperm samples totally). Following preparation of first-strand cDNA, relative quantification of the mature sequences of hsa-miR-30d-5p, hsa-miR-92a-3p, hsa-miR-132-3p and hsa-miR-204-5p was carried out by real-time PCR. The four miRNAs were selected based on our previous studies showing their constant detection in sperm of sires with diverse age, breed and fertility. The quantification cycles (Cq) of each miRNA was normalized using hsa-miR-30d-5p and hsa-miR-132-3p combined as reference genes. The relative expression of the four miRNAs was quantified using the ΔCq and 2- ΔΔCq methods (Livak and Schmittgen, Methods, 25:402-8, 2001). In HF samples, the fluorescent signal of miR-92a, miR-204, miR-132 and miR-30d was detected after Cq 22.34±1.10, 26.19±5.58, 26.69±2.20 and 28.05±3.32, respectively; the Cq values recorded in the LF group were 22.72±1.30, 23.23±1.92, 26.97±2.50 and 27.43±2.81, respectively. Expression of miR-204 was approximately 18-fold lower in the HF than in the LF group; this difference was significant only for miR-204 when applying a mixed-effects linear model with fertility group as fixed effect and the semen batch nested within sire as random effect (b=18.36±6.48, adjusted P=0.014). The expression of miR-92a, miR-132 and miR-30d did not differ between the two groups (adjusted P>0.05 in all cases). Concluding, our study showed the differential expression of the mature sequence of hsa-miR-204, a miRNA involved in chromatin condensation during spermiogenesis, in cryopreserved sperm of sires with diverse fertility status.