Application of Non-Destructive Methods: Biomarker Assays in Blood of White Stork (Ciconia ciconia) Nestlings

Abstract

White stork (Ciconia ciconia) nestlings can provide quantitative information on the quality of the surrounding environment by indicating the presence of pollutants, as they depend on locally foraged food. This study represents the first comparison of biomarkers in two fractions of white stork nestling blood: plasma and S9 (the post-mitochondrial fraction). The aim of this study was to evaluate acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST), and glutathione reductase (GR), as well as to establish a novel fluorescence-based method for glutathione (GSH) and reactive oxygen species (ROS) detection in plasma and S9. Considering the enzymatic biomarkers, lower variability in plasma was detected only for AChE, as CES, GST, and GR had lower variability in S9. Enzyme activity was higher in plasma for AChE, CES, and GST, while GR had higher activity in S9. Regarding the fluorescence-based method, lower variability was detected in plasma for GSH and ROS, although higher GSH detection was reported in S9, and higher ROS was detected in plasma. The present study indicated valuable differences by successfully establishing protocols for biomarker measurement in plasma and S9 based on variability, enzyme activity, and fluorescence. For a better understanding of the environmental effects on nestlings’ physiological condition, biomarkers can be measured in plasma and S9.

Blood biomarkers in white stork (Ciconia ciconia) nestlings show different responses in several areas of Croatia

“…All nestlings were between 6 and 8 weeks old. Nondestructive methods were used to obtain the blood samples and two blood fractions were prepared for efficient evaluation of nestlings' condition (Bjedov et al, 2021). Blood samples were collected with a sterile 5 mL syringe and 0.8 mm needle.…”

“…Plasma (supernatant) aliquots were distributed to the new sterile 2 mL tubes and stored at –80°C until analysis. Pellets were prepared as described in Bjedov et al (2021). In brief, the pellet was suspended in 5 mL of 0.1 M sodium phosphate buffer (pH 7.2) and sonicated for cell disruption, following centrifugation at 9000 g for 20 min at 4°C to acquire the postmitochondrial fraction (S9).…”

“…In our previous study (Bjedov et al, 2021), we have shown that blood sampled from white stork nestlings during a regular ringing scheme can be used for the measurement of different biomarkers in two types of blood samples. Considering the information that can be obtained from biomarker responses, such as health status or potential exposure to harmful substances, the aim of this study was to investigate biomarker responses to environmental pollutants in avian blood.…”

“…Plasma (supernatant) aliquots were distributed to the new sterile 2 mL tubes and stored at -80°C until analysis. Pellets were prepared as described in Bjedov et al (2021). In brief, the pellet was…”

“…Sex was determined from S9 according to Begović et al (2017) with modifications described in Bjedov et al (2021), based on CHD gene amplification (Fridolfsson & Ellegren, 1999).…”