A Combined Flow Cytometric Semen Analysis and miRNA Profiling as a Tool to Discriminate Between High- and Low-Fertility Bulls

Abstract

Predicting bull fertility is one of the main challenges for the dairy breeding industry and artificial insemination (AI) centers. Semen evaluation performed in the AI center is not fully reliable to determine the level of bull fertility. Spermatozoa are rich in active miRNA. Specific sperm-borne miRNAs can be linked to fertility. The aim of our study is to propose a combined flow cytometric analysis and miRNA profiling of semen bulls with different fertility to identify markers that can be potentially used for the prediction of field fertility. Sperm functions were analyzed in frozen-thawed semen doses (CG: control group) and high-quality sperm (HQS) fraction collected from bulls with different field fertility levels (estimated relative conception rate or ERCR) by using advanced techniques, such as the computer-assisted semen analysis system, flow cytometry, and small RNA-sequencing. Fertility groups differ for total and progressive motility and in the abnormality degree of the chromatin structure (P < 0.05). A backward, stepwise, multiple regression analysis was applied to define a model with high relation between in vivo (e.g., ERCR) and in vitro (i.e., semen quality and DE-miRNA) fertility data. The analysis produced two models that accounted for more than 78% of the variation of ERCR (CG: R2 = 0.88; HQS: R2 = 0.78), identifying a suitable combination of parameters useful to predict bull fertility. The predictive equation on CG samples included eight variables: four kinetic parameters and four DNA integrity indicators. For the HQS fraction, the predictive equation included five variables: three kinetic parameters and two DNA integrity indicators. A significant relationship was observed between real and predicted fertility in CG (R2 = 0.88) and HQS fraction (R2 = 0.82). We identified 15 differentially expressed miRNAs between high- and low-fertility bulls, nine of which are known (miR-2285n, miR-378, miR-423-3p, miR-191, miR-2904, miR-378c, miR-431, miR-486, miR-2478) while the remaining are novel. The multidimensional preference analysis model partially separates bulls according to their fertility, clustering three semen quality variable groups relative to motility, DNA integrity, and viability. A positive association between field fertility, semen quality parameters, and specific miRNAs was revealed. The integrated approach could provide a model for bull selection in AI centers, increasing the reproductive efficiency of livestock.

Identification of differentially expressed mRNAs and miRNAs in spermatozoa of bulls of varying fertility

“…Knockdown of FoxO1, FoxO2 and FoxO4 genes has been shown to impair mouse preimplantation embryonic development ( 66 ). In the current study, it is difficult to decipher the collective actions of the differentially expressed miRNAs, but from previous studies it is likely that these miRNAs have an effect on early embryo development, prior to embryonic genome activation ( 23 , 67 ). Further analyses, carried out on embryos generated with the semen from HF and LF bulls, is needed to corroborate this.…”

“…Capra et al ( 22 ) identified 83 differentially expressed miRNAs between bulls with high and low motile spermatozoa, with these miRNAs targeting biological pathways related to apoptosis. Recently, Turri, et al ( 23 ) reported 13 differentially expressed miRNAs between HF and LF with miR-423-3p highly expressed among LF bulls, which is known to be associated with severe asthenozoospermia in human spermatozoa. In the same study, miR-191 had increased expression in HF bulls and has previously been positively associated with the fertilization rate of blastocysts ( 24 ).…”

Investigation of Sperm and Seminal Plasma Candidate MicroRNAs of Bulls with Differing Fertility and In Silico Prediction of miRNA-mRNA Interaction Network of Reproductive Function

“…Sire conception rate showed a positive correlation with relative sperm volume shift [ 35 ] and acrosome reacted sperm at 5 h post-thaw [ 34 ], no correlation with hypoosmotic swelling [ 35 ], negative correlation with lipid peroxidation [ 171 ], sperm DNA fragmentation, and plasma membrane integrity [ 172 ]. Turrri et al (2021), observed a positive association of field fertility (estimated relative conception rate), semen quality parameters (sperm kinetics and DNA), and specific miRNAs (15 differentially expressed miRNAs (including nine known miR-2285n, miR-378, miR-423-3p, miR-191, miR-2904, miR-378c, miR-431, miR-486, miR-2478)) between high- and low-fertility bulls [ 21 ].…”

“…MicroRNAs are highly stable in biofluids. Expression of miRNAs is detectable in sperm and seminal plasma of all animal species [ 17 , 18 , 19 , 20 , 21 , 22 ]. The miRNA expression is greater in sperm compared to seminal plasma and follows a similar expression trend [ 9 , 10 ].…”

Significance and Relevance of Spermatozoal RNAs to Male Fertility in Livestock

“…Spermatozoal miRNAs have been reported in healthy bulls and those differeing in their spermatozoal motility or fertility ( Table 3 ). The whole miRNA profiling of bull spermatozoa through deep sequencing identified 2022 miRNAs and included a large number of isomiRs across different species ( Turri et al, 2021 ). RNA-seq analysis of high fertility and low fertility bulls reported nine DE miRNAs (miR-2285n, miR-378, miR-423-3p, miR-19, miR-2904, miR-378c, miR-431, miR-486, and miR-2478) in which miR-2285n, miR-378, and miR-486 were observed to have altered expression levels between low and high motile sperm.…”