# Alternative Methodology for Cortisol Evaluation Before and After Sheep Shearing Using Raman Spectroscopy: A Feasibility Study

## TL;DR

The study shows Raman spectroscopy can detect cortisol changes in sheep serum before and after shearing, offering a faster alternative to traditional methods.

## Contribution

This is the first feasibility study demonstrating Raman spectroscopy's potential for cortisol evaluation in sheep serum.

## Key findings

- Raman spectroscopy detected cortisol in sheep serum with results matching ELISA measurements.
- Cortisol levels increased after shearing, as observed through Raman band intensity changes.
- The technique showed a positive correlation between Raman and ELISA assessments across all time points.

## Abstract

Raman Spectroscopy is a rapid and alternative methodology applied in many biological tissues for the assessment of their compounds. The aim of the present study was to compare the serum cortisol levels assessed by means of an ELISA kit and Raman Spectroscopy. To do this, serum was collected in sheep subjected to shearing before and 5 and 60 min after the procedure; this is well known to be stressful for these animals. The obtained results demonstrate the feasibility of Raman spectroscopy analysis for the identification of cortisol in sheep serum. Further studies are necessary to verify its applicability to other physiological parameters in this species.

This research proposed a feasibility study for a qualitative and semiquantitative analysis of sheep serum using Raman Spectroscopy analysis as an alternative to standard methodologies. Raman Spectroscopy was used to obtain information about molecular vibrations that can provide information about the behavior of specific variations in the protein network. This study was conducted during the traditional vernal shearing procedure in Sicilian sheep breeding. Twenty female sheep were randomly chosen from a one-hundred Comisana-bred flock habituated to the handling required for shearing and venipuncture. Animals had a mean body weight of 52.35 ± 4.55 kg, were aged between 2 and 3 years old, and were clinically healthy with no evidence of disease and free from internal and external parasites. All animals were shorn on the same day by hand using traditional shearing scissors in a 15 m × 10 m pen. The animals were released into an adjacent pen at the end of the shearing procedure. For each animal, blood samples were collected through jugular venipuncture into a vacutainer tube with a clot activator (Terumo Corporation, Japan) immediately before and 5 and 60 min after the end of the shearing procedure. On the obtained sera, ELISA and Raman spectroscopy analyses were performed to evaluate cortisol concentrations. The band area corresponding to the cortisol vibration mode was identified in the 1300–1366 cm−1 band. The Raman spectra obtained during the various protocol data points showed the same trend, with differences in the intensity of the band area 1300–1366 cm−1. A positive correlation was found between ELISA and Raman assessment in all experimental conditions. The obtained results demonstrate that Raman spectroscopy analysis could be a suitable tool for biomolecule identification. This study demonstrated that this technique provides useful information for understanding sheep responses to stress induced by management conditions.