Sex-specific Epitopic Peptide Synthesis, Purification and Characterisation to Target Bovine Spermatozoa

Abstract

The present research was conducted during July, 2025 to September, 2025 at Division of Biochemistry, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India which mainly focused on the identification and synthesis of sex-specific peptide sequences associated with bovinespermatozoa. Initially, an extensive review was carried out using data obtained from various proteomic studies on bovine spermatozoa, where differentially expressed as well as specifically expressed proteins had been identified and systematically listed. Based on these earlier proteomic findings, SCAMP1 was selected as a potential candidate because it had been reported to be Y-chromosome-specific. To further investigate its immunogenic potential, epitope prediction and peptide design were performed using several bioinformatic tools, including DeepTMHMM and SEMA 2.0. These computational approaches helped in identifying suitable peptide regions with desirable antigenic and structural characteristics. Subsequently, the selected peptide sequence was synthesized through Fmoc chemistry-based solid-phase peptide synthesis employing the Multiple Antigen Peptide (MAP) format, which enhances peptide immunogenicity and stability. After synthesis, purification of the peptide was performed using High-performance Liquid Chromatography (HPLC) to obtain a highly purified product. Furthermore, structural characterization of the synthesized peptide was carried out using Circular Dichroism (CD) spectroscopy in order to evaluate its secondary structural conformation and confirm the integrity of the synthesized peptide. The present study aimed to investigate the potential of a sex-specific epitopic peptide and evaluate its possible application in the development of a novel sperm sexing technology. By identifying and characterizing sex-specific peptide epitopes, the study sought to provide a targeted and efficient approach for distinguishing X- and Y- spermatozoa.