The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting

Abstract

Background: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRR_conv and NRR_ss, respectively) was recorded for each bull.

Results: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRR_ss but not to NRR_conv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRR_ss.

Conclusions: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm.

Keywords: bull fertility; miRNA; microRNA; sex-sorted sperm; small RNA-seq.

Fig. 1

Number of total reads in unsorted sperm samples of 18 bulls. The dark blue bar fraction represents the part of total reads with length of 18–30 nucleotides. Bulls A to I and bulls J to R showed low and high fertility after artificial insemination with X-bearing cryopreserved sperm, respectively

Fig. 2

Tukey-style boxplots for the counts per million reads (a) and relative abundancy (b) of the top 10 miRNAs detected in unsorted sperm samples obtained from low- (LF) and high-fertile (HF) bulls

Fig. 3

Correlation circle for the 85 identified sperm miRNAs (a) and visualization of the sperm samples obtained from low- (LF) and high-fertile (HF) bulls (b), plotted against the first two principal components (Dim 1 and 2, respectively)

Fig. 4

Robust regression lines of five miRNAs predicting the non-return rate for sex-sorted sperm (a); Tukey-style boxplots for the expression levels of the five miRNAs in the high-and low-fertility group (b). a. Robust regression lines with 95% confidence intervals (grey shaded area) for single predictors (predicted NRR_ss values plotted against the observed expression levels of single miRNA, when all other predictors are kept constant at their mean value) are presented. Plotted points represent the observed NRR_ss values; red circles indicate bulls identified and treated by the robust regression model as outliers in regard to their sperm miRNA profile. NRR_ss, 56-day non-return rate for sex-sorted sperm; cpm, count per million reads b. LF, low-fertility group; HF, high-fertility group

Fig. 5

Standardized b coefficients for two robust regression models describing the relation between the expression levels of five sperm miRNAs and the 56-day non-return rate of 15 bulls recorded after artificial insemination with unsorted (orange) and sex-sorted (blue) cryopreserved sperm, respectively. Transparent points represent the mean of the standardized coefficient b, the horizontal line the 95% confidence intervals and the thicker part of the line the 90% confidence intervals of the estimated coefficients

Fig. 6

The top 20 gene ontology enriched clusters (biological processes) for the predicted targets of the two differentially expressed miRNAs (miR-9-5p, miR-10a-5p; top panel), and four out of five robust regression predictors (miR-34c, miR-342, miR-106b-5p, miR-92a; bottom panel), colored for P value. log10(P), P value in log base 10