167 Exploring the potential of Raman spectroscopy to discriminate boar semen samples during storage

Dependence on liquid semen is a burden in the hog industry. Despite the generally applied thresholds for breeding purposes, the industry still faces challenges related to individual boar variations and the gradual decline in semen quality during prolonged storage. There is a critical need for innovative approaches for the early prognosis of sperm with divergent motility profiles during storage. Besides providing the advantages of precision, repeatability, noninvasiveness, and little or no sample preparation, Raman spectroscopy allows for assessing biochemical subtleties and changes undetectable by the routine semen analysis. This study used Raman spectroscopy to evaluate sperm samples with differential ability to withstand prolonged storage. Extended semen doses of boars (n = 26) were obtained from a commercial breeding company (Prestage Farms, MS) and transported to the laboratory. Semen doses were immediately fractionated, and aliquots were stored at 16–18°C for 10 days post-collection. Every day, sample aliquots of extended semen were subjected to sperm motility analysis using the Computer-Assisted Sperm Analyzer (CASA; CEROS), followed by the Raman spectroscopic analysis (785 nm laser excitation for 20 s). Thereafter, samples were centrifuged (5 min; 900g) to collect the supernatants, which were replaced by an equal volume of phosphate-buffered saline solution to resuspend the resulting sperm pellets. Both sample types were also subjected to spectroscopic analysis. Semen doses were obtained in four independent collection days (Day 0), and samples displaying the highest (Good) and lowest (Poor) motility and normal morphology on Day 7 were investigated. Data were analysed (analysis of variance repeated measurements and Tukey post hoc test) and expressed as mean ± s.e.m. A P < 0.05 indicates a significant difference. On Day 0, samples displayed averages of 74.2 ± 3.9% total motility, 33.3 ± 7.4% progressive motility, and 86.1 ± 2.1% normal morphology as measured with CASA. However, on Day 7, the heterogeneity among samples’ motility decline allowed discrimination between Good- and Poor-quality semen based on total motility (74.7 ± 5.2% vs 14.1 ± 6.1%), progressive motility (38.6 ± 7.0% vs 2.0 ± 1.0%), and normal morphology (81.1 ± 2.6% vs 62.6 ± 3.6%). Interestingly, sperm motility and morphology parameters remained unchanged (P > 0.05) up to Day 7 in Good samples, while the declines appeared as on Day 3 (motility: 52.8 ± 12.9%; progressive: 24.0 ± 7.1%; morphology: 75.9 ± 3.4%) in the Poor samples. Raman spectra of extended semen of Good and Poor samples exhibited different profiles, with marked differences in the 1600 and 2400 cm⁻¹, on Day 0 and Day 7 of storage. Likewise, the spectra of the corresponding supernatants showed different profiles with major differences in the 1600–2000 cm⁻¹ domain, usually assigned to C=O bonds vibrations in nucleic acids, esters, and fatty acids, and in the 2380–2650 cm⁻¹ domain, corresponding to CH₃ vibrations in lipids and nucleic acids. Findings provide evidence of the potential of Raman spectroscopy to aid in the robust assessment of semen resilience to cold storage. Ongoing studies are validating Raman spectroscopy as a new tool to improve boar semen storage.

Research was funded by USDA-ARS grant #6066–31320–017–000D.