316 FOURIER TRANSFORM INFRARED MICROSPECTROSCOPY: A NEW APPROACH FOR MOLECULAR PROFILE ANALYSIS OF SPERMATOZOA AND SEMINAL PLASMA

Abstract

Fourier transform infrared (FT-IR) spectroscopy is a potential tool for noninvasive optical tissue diagnosis. Applications of this technique for non-clinical purposes have increased since it has been shown that it can also provide the identification of functional groups, bonding types, and molecular conformations. Moreover, the spectral bands are molecule-specific and provide direct information about the biochemical composition. However, the exploitation of FT-IR spectroscopy for reproductive studies is still limited. The aim of this study was to determine molecular profiles of spermatozoa and seminal plasma from different species to define important peaks present in the natural tissues. The study was based on fresh semen samples from 4 different species (human, bovine, ovine, and porcine). Semen samples were collected and evaluated for concentration and motility parameters. After that, samples were centrifuged and the seminal plasma was transferred to another tube. Spermatozoa were washed three times in PBS and 5 × 106 cells were resuspended in 50 μL of deionized water. Samples of 2 μL from spermatozoa and seminal plasma were placed separately on a gold slide and left to dry for 15 min in air. Spectral data were collected on a FT-IR spectrometer (Model 610; Varian Inc., Palo Alto, CA, USA). The absorbance spectra were acquired in reflectance mode at a spectral resolution of 2 cm^-1 with 400 scans co-added. Apodization was performed using a triangular function. The system enabled spectra to be acquired from a sample area of approximately 200 μm² in about 2 min. The preliminary tentative vibrational bands assignment was performed based on literature. Spectral differences among species were statistically validated with principal components analysis and clustering. The main spectral differences among spermatozoa spectral bands were observed in the DNA/RNA (A, B, and Z conformations), glycogen, lipids, fatty acids, cholesterol, and phospholipids vibrational bands. For seminal plasma, main spectral differences were found at the 1500-1700 cm^-1 spectral region assigned to protein secondary structure (amide I and amide II). Increasing a-helix, fi-sheet, fi-turn bands assignments suggests the presence of fragmented protein in human, ovine, and porcine seminal plasma spectra. These results suggest compositional similarity among these 3 species compared to the bovine. In conclusion, FT-IR spectroscopy could provide relevant information related to spermatozoa and seminal plasma physiology useful in improving freezing methods and fertility monitoring. More studies will be needed to establish the relationship between spectral features and physiological processes.