Fourier Transform Infrared Spectroscopy to Measure Cholesterol in Goat Spermatozoa

Sperm cryopreservation produces a series of physicochemical phenomena that negatively impact the function and structure of spermatozoa, including the mobilization of cholesterol from the plasma membrane. The use of attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR) may be useful to measure the cholesterol efflux in goat spermatozoa. Therefore, the objective of this study was to standardize the use of ATR-FTIR to measure the efflux of cholesterol in goat spermatozoa. Standardization of the technique was carried out in three stages: (i) determination of the appropriate sperm concentration to detect cholesterol in the FTIR spectrum; (ii) determination of the minimum percentage of viable spermatozoa required to observe at least five spectral bands in common with pure cholesterol; (iii) assessment of cholesterol removal in frozen-thawed spermatozoa. Possible differences in the areas of the spectral bands were compared by one-way ANOVA. Nineteen spectra were obtained: pure cholesterol, sperm transport medium, five different sperm concentrations, and ten live/dead sperm proportions (heat and cold-killed). The lowest sperm concentration at which spectral bands were clearly identified was 13 × 10⁶ sperm/mL. Regarding viability, the cut-off value was 50%: higher values produced spectral bands clearly detectable, whereas in smaller values, the band's areas decreased sharply, making it difficult to quantify them. Five areas of the cholesterol bands decreased in thawed samples compared to fresh spermatozoa; an increase in the proportion of frozen-thawed sperm showing Merocyanine brilliant pattern, indicative of high fluidity, as well as an increase in the proportion of CTC AR pattern, indicative of acrosome reaction, support those results. In conclusion, ATR-FTIR is a useful technique for identifying the movement of cholesterol in goat buck spermatozoa.