# Fourier Transform Infrared Spectroscopy to Measure Cholesterol in Goat Spermatozoa

## TL;DR

This study shows that ATR-FTIR can effectively measure cholesterol changes in goat sperm, especially after freezing and thawing.

## Contribution

The study standardizes ATR-FTIR for cholesterol quantification in goat spermatozoa under various conditions.

## Key findings

- The optimal sperm concentration for detecting cholesterol via ATR-FTIR is 13 million sperm/mL.
- A viability threshold of 50% is needed for reliable cholesterol spectral band detection.
- Cholesterol levels decrease in thawed sperm compared to fresh samples, as shown by spectral band area changes.

## Abstract

This study standardized the use of attenuated total reflection–Fourier-transform infrared spectroscopy (ATR-FTIR) to measure cholesterol in goat buck sperm. Cholesterol is a component of the sperm plasma membrane that is removed during both physiological (capacitation, to be fertile) and non-physiological (cryopreservation) conditions. For this, three experiments were carried out: (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. The lowest sperm concentration at which spectral bands were clearly identified was 13 million sperm/mL; regarding viability, the cut-off value was 50%. Five areas of the cholesterol bands decreased in thawed compared to fresh spermatozoa. In conclusion, FTIR is a useful technique to quantify the cholesterol efflux in 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.