Proteomics Evaluation of Semen of Clinically Healthy Beagle-Breed Dogs

Simple Summary Proteomics aims to identify proteins present in a sample and to study their expression during various physiological or pathological conditions. The proteome includes all proteins present in a cell or a tissue at any given time; this takes into account all post-translational modifications that occur, and is highly dynamic. The present work used high-throughput technologies to study the proteome of the semen of dogs. In total, 42 proteins were identified in the semen sperm-rich fraction and 43 proteins in the semen prostatic fraction. In general, the proteins identified are involved mostly in supporting spermatozoan maturation, survival and motility, enhancing the reproductive performance of male animals. Future work can focus on the quantification of proteins identified in semen and compare findings with results in samples from animals with suboptimal fertility. The findings can provide a potential for proteomics examination of semen as a tool in se-men evaluation. This can be particularly useful in stud animals, also given its advantage as a non-invasive method. Abstract The objectives of the present work were to evaluate the semen of dogs by means of proteomics methods and to compare with proteomics results of the blood of the animals, in order to increase available knowledge on the topic and present relevant reference values for semen samples. Semen samples were collected from five Beagle-breed dogs. Reproductive assessment of the animals by means of clinical, ultrasonographic and seminological examinations confirmed their reproductive health. The sperm-rich fraction and the prostatic fraction of semen were processed for proteomics evaluation. LC-MS/MS analysis was performed by means of a LTQ Orbitrap Elite system. The technology combines high separation capacity and strong qualitative ability of proteins in biological samples that require deep proteome coverage. Protein classification was performed based on their functional annotations using Gene Ontology (GO). In blood plasma, semen sperm-rich fraction, and semen prostatic fraction, 59, 42 and 43 proteins, respectively, were detected. Two proteins were identified simultaneously in plasma and the semen sperm-rich fraction, 11 proteins in plasma and the semen prostatic fraction, and three proteins in the semen sperm-rich and prostatic fractions. In semen samples, most proteins were related to cell organization and biogenesis, metabolic processes or transport of ions and molecules. Most proteins were located in the cell membrane, the cytosol or the nucleus. Finally, most proteins performed functions related to binding or enzyme regulation. There were no differences between the semen sperm-rich fraction and prostatic fractions in terms of the clustering of proteins. In conclusion, a baseline reference for proteins in the semen of Beagle-breed dogs is provided. These proteins are involved mostly in supporting spermatozoan maturation, survival and motility, enhancing the reproductive performance of male animals. There appears potential for the proteomics examination of semen to become a tool in semen evaluation. This analysis may potentially identify biomarkers for reproductive disorders. This can be particularly useful in stud animals, also given its advantage as a non-invasive method.