168 Proteomic profiling and functional annotation of indigenous Zulu ram seminal plasma

The South African indigenous Zulu sheep is an important breed, particularly to communal farmers, owing to its desirable traits such as disease tolerance and excellent adaptation to harsh environments. However, the breed remains undesirable due to low reproductive performance, which may be attributed to male fertility. Traditional semen quality tests provide limited information about the potential fertility of rams. Ram seminal plasma (SP) is a highly concentrated protein-rich fluid and has numerous extracellular vesicles. A proteomic analysis of SP can help identify functionally important proteins and their role in the regulation of various processes of fertilization. The purpose of this study was to thoroughly investigate the fertility and reproductive capabilities of this breed through rigorous proteomic profiling and functional annotation of the SP proteins. Semen was collected bi-weekly from six Zulu rams for 4 months using an electro ejaculator at 3–6 V for 3 s at 7-s intervals. The total sperm motility evaluated ranged from 80% to 99%, and the sperm concentration ranged from 29.9 to 81 m mL⁻¹. Eight seminal plasma samples from each ram were harvested by centrifugation and prepared for mass spectrometry. An Evosep One liquid chromatography system coupled to an AB Sciex 6600 TripleTOF mass spectrometer (AB Sciex) was used to analyze 500 µg of SP peptides. R package protti was used to statistically analyze differential expression and identify protein stability and variation in semen ejaculates collected on different days. Rank intensities were used to discover the abundance of proteins, and Uniprot and ShinyGO were used for functional enrichment analysis. The study identified a total of 754 SP proteins and profiled the 10 most and least abundant proteins. Microphage migration inhibitory factor (MIF), which is involved in sperm flagellar beating, was among the highly abundant proteins. It was followed by minichromosome maintenance complex component 3 associated protein (MCM³), which can be used as a marker for cell proliferation given its involvement in initiation and regulation of DNA replication. The low abundance of GTPase and IQGTPase indicated high fertility because their high abundance is associated with male sterility. The variation in stability of proteins from SP of ejaculates collected on different days was observed from the differentially expressed proteins mostly on the eighth collection. The downregulated expression of membrane metalloendopeptidase (MME) may indicate increased levels in testosterone, while the reduced expression of heat shock protein family A (HSp70) member 4 like indicates reduced stress levels. In conclusion, the high abundance of proteins such as MIF and MCM³ and low abundance of others, such as GTPase and IQGTPase, can be used as fertility biomarkers because they indicate high fertility of Zulu rams. Zulu rams acclimatize to the process of semen collection overtime as indicated by reduced HSp70. The downregulation of MME may indicate increased testosterone secretions and reduced degradation of peptides required for the maintenance of sperm function.