161 Proteomics analysis of buffalo ejaculates having slow, normal, and fast nondirectional motile sperm

Murrah buffalo bull ejaculates were divided into normal, slow, and fast nondirectional categories on the basis of subjective analysis by phase contrast microscope. Protein was extracted from sperm pellet samples. Label-free proteomic quantification was done by mass spectrometric analysis using EASY-nLC 1200 system (Thermo Fisher Scientific) coupled to a Thermo Fisher Q Exactive Plus mass spectrometer equipped with a nanoelectrospray ion source. The samples were processed, and the RAW files generated were analyzed with Proteome Discoverer (v2.4) against the Uniprot Bovine proteome database. For Sequest and Amanda search, the precursor and fragment mass tolerances were set at 10 ppm and 0.02 Da, respectively. The protease used to generate peptides (i.e. enzyme specificity) was set for trypsin/P (cleavage at the C terminus of “K/R: unless followed by “P”) along with maximum missed cleavages value of two. Carbamidomethyl on cysteine as a fixed modification and oxidation of methionine were considered variable modifications for the database search. A total of 1645 proteins were detected in buffalo spermatozoa. Out of 1645 proteins, 417 proteins were differentially expressed in the slow motile group compared with the normal motile group, while only 331 proteins were differentially expressed in fast motile group relative to the normal motile group. Among differentially expressed proteins, 157 proteins in the slow motile group and 136 proteins in the fast motile group were high in abundance from the normal motile group. In contrast, 258 proteins in the slow motile group and 196 in the fast motile group were low in abundance relative to the normal motile group. To identify the differentially expressed proteins exclusive to the slow and fast motile groups, comparative Venn diagrams were used. The results showed that 242 proteins were exclusively expressed in the slow motile group, 156 proteins were exclusively expressed in the fast motile group, and 175 proteins were common to both groups. Among the differentially upregulated proteins, 104 were exclusively expressed in the slow motile group and only 83 in the fast motile group. Among differentially downregulated proteins, 146 proteins were exclusively found in the slow motile group and while 81 proteins in fast motile group. Proteomic analysis generated an important panel of candidate proteins that may be responsible for different motility pattern of buffalo spermatozoa.