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MicroRNA sequences (miRNA) are small RNAs 19 to 22 nucleotides in length that play a crucial role during mammalian development and disease. They are found in plants and animals and regulate the expression of protein-coding genes. Mechanisms regulating gene expression during gamete and embryo development are critical for setting the stage in later development; however, very little is known about the roles of miRNAs in male gametes (spermatozoa). Therefore, the objective of this study was to profile miRNA populations in spermatozoa collected from high and low fertility bulls. Frozen–thawed straws containing spermatozoa from one low and one high fertility bulls were purified using a Percoll gradient (which removes somatic cells) followed by 3 washing steps in PBS. Total RNA were isolated from pelleted spermatozoa using Trizol (3 independent isolations for each bull, total of 6 samples). RNA samples were quantified (NanoDrop Spectrophotometer, Thermo Scientific, Wilmington, DE) and verified for potential somatic cells (Agilent BioAnalyzer, Agilent, Foster City, CA) and DNA (No-RT-PCR) contaminations. Samples devoid of any contaminations went through an enrichment process of miRNA, consisting of dephosphorylation (CIP) and labeling (pCp-Biotin) of the 3′-end of miRNA, followed by their purification (BioSpin6, Bio-Rad, Hercules, CA). The purified miRNAs were analyzed by a miRNA microarray containing approximately 30 000 probes from human, rat, mouse, and other organisms (Ambion/Affymetrix DiscovArray, Asuragen, Austin, TX). The results showed that roughly 48% of total miRNA probe sets (14 215) were successfully analyzed (P2 and t-test P< 0.001). The roles of these miRNAs are not yet identified because the annotations are unavailable. Nevertheless, our preliminary results suggest that bull spermatozoa are rich in miRNAs, which could play important roles during fertilization and embryo development. Future studies will be aimed at identifying potential molecular mechanisms by which these 7 miRNAs might regulate bovine embryonic development.This study was funded by the Mississippi Agricultural and Forestry Experiment Station and Alta Genetics Inc. [ABSTRACT FROM AUTHOR]

The reproductive performance of a herd is the biggest factor affecting production and product quality of livestock. Thus, a decline of male fertility represents a dramatic economic loss in beef and dairy industries. Caused by molecular defects in the spermatozoa, uncompensatory infertility is a current challenge for the cattle industry, because even with normal sperm morphology, motility, and number, fertility of bulls is still sub-par. Thus, the objective of this study was to determine the global proteome of spermatozoa collected from bulls with different fertility and study the proteins playing a role in uncompensatory infertility. We performed difference gel electrophoresis (2D-DIGE) using cryopreserved sperm from a total of 6 bulls. Spermatozoa were thawed, purified by percoll gradient, and washed in PBS solution. For each bull, a pellet of 100 million sperm cells was resuspended in the 2D-DIGE labeling buffer, and the total protein was quantified. Cell lysates were separately labeled with CyDye DIGE Fluor dyes (and reciprocally with different dyes) and multiplexed in pairs on 3 gels. The samples were focused according to their isoelectric point through an Immobiline DryStrip, pH4-7, using a IPGphor 2, followed by separation on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels (21.5–200 kDa) using a DALTsix. The resulting gels were scanned, and their topographic digital maps were used for algorithmic spot matching, background normalization, spot differences quantification, and elimination of artifacts (DeCyder 2-D Differential Analysis Software, GE Healthcare Life Sciences, Piscataway, NJ, USA). The results showed between 2600 and 2800 proteins with high confidence. Compared to the high-fertile bulls, 30 proteins were increased, and 27 were decreased in the low-fertility bulls within a 2-fold range. The largest significant increase and decrease were 3.97- and 2.4-fold, respectively. The identification of these differentially represented proteins is in progress. However, our results provided a panoramic view of sperm proteome from bulls of different fertility and thus paved the way for research on mechanisms of uncompensatory bull infertility. [ABSTRACT FROM AUTHOR]