Paper Title
“HIGH ABUNDANCE OF DLD PROTEIN IN SPERMATOZOA LEADING TO INCREASED ROS PRODUCTION AND EARLY CAPACITATION IN BUFFALO BULL SPERMATOZOA”.

Abstract
The water buffalo (Bubalus bubalis) is an indispensable part of the Indian dairy sector. Many instances, farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential. Hence, it becomes a paramount importance to predict precisely the fertility status of bulls before performing AI.It is well understood that mature bovine sperm are terminally differentiated cell which are transcriptionally and translationally quiescent. However, these spermatozoa are well equipped and carry pre-synthesized proteins as final products to accomplish the fertilization event in female reproductive tract. Many conventional approaches are currently available to assess the fertilizing potential of bull spermatozoa. The high throughput proteomics offer far superior strategy than that of other "omics" approaches for identifying the fertility biomarkers in sperm. Sperm proteins play a key role in maintaining their structure, survival and fertilizing capability in the female reproductive tract (FRT). The question arises in this context of how the differential abundance of the protein in spermatozoa regulates the bull fertility. Additionally, it would be interesting to identify the vital proteins in buffalo spermatozoa and elucidating their possible mode of action via different pathways regulating the key functions of spermatozoa. In this study, the global proteomic profile of high fertile (HF) and low fertile (LF) buffalo bull spermatozoa was established using a high-throughput LC-MS/MS technique to find out the differential abundant proteins involved in fertility. Mass spectrometry (MS) data analyses revealed that a total of 1385 proteins were identified in buffalo spermatozoa, out of which HF and LF groups possessed 1290 and 1097 proteins. Among these, 1002 were common to both the groups, categorized as differentially abundant proteins (DAPs), while 288 and 95 proteins were unique to HF and LF groups respectively. Further analysis of data, we identified 553 proteins as significantly differentially abundant between the HF and LF spermatozoa. In HF spermatozoa, nearly 211 proteins were high abundant (log fold change or Fc ≥ 2) while 342 proteins were low abundant (log Fc <0.5). We performed Gene Ontology and Pathway enrichment analysis of identified proteins using DAVID functional annotation tool. From the list of DAPs, the three most promising proteini.e DLD were shortlisted based on high fold change in mass spectrometry data reflecting the corresponding peptide matches. More importantly, these proteins were selected based on their involvement in the major pathways and vital role in regulating the sperm fertilizing capacity. The mechanism of action of DLD protein in buffalo spermatozoa was assessed by utilizing the specific antagonist for target proteins under in-vitro sperm model. To find out the functional significance of DLD protein in fertility we used MICA (2-methoxyindole-5-carboxylic acid) inhibitor in dose and time dependent manner. Based on data interpretation, the current leads demonstrate that DLD protein regulates ROS generation, motility, capacitation, and acrosome reaction in buffalo spermatozoa. In addition, the findings of this study convincingly reveal that excess amount of DLD protein caused an early capacitation and acrosome reaction in spermatozoa which in turn reduce the fertilizing potential of the buffalo spermatozoa. The DLD protein remained abundant in low fertile bulls, and it was responsible for producing elevated level of ROS to induce the early capacitation process in spermatozoa. In conclusion, the DAPs identified in this study are strongly associated with sperm functions, hence, they can be used as ideal and potential protein candidates for predicting buffalo bull fertility.