Spermatozoa were isolated from the seminal fluid using washing (wash), post-capacitation (POS), and swim-up (SU) techniques, followed by analysis through Surface-Enhanced Raman Spectroscopy (SERS). Density gradient and swim-up methods were applied to 35 semen samples to validate sperm quality. The resulting spectra showed notable variations at 408 cm−1 (S–S stretch attributed to lysozyme) and 728 cm−1 (associated with DNA alterations and methylation). These spectral markers were incorporated into partial least squares discriminant analysis (PLS-DA) models to distinguish among sperm populations prepared by different methods. One PLS-DA model differentiated wash from POS and SU, attaining 86% sensitivity and 91% accuracy. Another model distinguished between POS and SU, achieving 77% sensitivity and 74% accuracy. The combined use of SERS and multivariate analysis offers a promising alternative for assessing sperm quality, supported by motility assessments in 35 validated samples. This approach could enhance both the accuracy and efficiency of reproductive diagnostics.
