Experimental Design
This observational study was carried on in November 2020 during an OOS training that included a theoretical part ashore, a full training day of inshore sailing, and three days of non-stop OOS roughly between the latitudes of Gibraltar and Lisbon. The crew sailed in a Class 40 (ITA 84) racing yacht and, during the navigation, was divided into two groups alternating rest and duty shifts every 3 hours. Figure 1 depicts the study protocol and samplings. Urine and saliva samples and anthropometric measurements were obtained ashore during the theoretical part (PRE) and after the navigation (POST). Further, two urine samples were obtained during the single day of inshore navigation (Training) and three times a day during OOS (Sailing).
Subjects
This study involved six male sailors: the skipper (SF) - a professional sailor with experience in ocean solo races - and five recreational sailors with good expertise in seamanship. The subjects were all healthy, and their characteristics are reported in Table 1.
Navigation
The offshore navigation lasted three days, during which the crew sailed into the ocean for a total of 420 miles, with a top speed of 14.89 kn.
During the first day and night, the atmospheric conditions were challenging. Swell of 3/3.5 meters Significant Wave Height (SWH) from NW and wind from 5 to 15kn from S-SE resulted in a boat's inconvenient motion. During the day, the wind increased between 25 and 40kn in gusts as sailors encountered two significant squalls and had to flee downwind. During the night, sailors were forced to maintain a 70° True Wind Angle (TWA) sailing upwind to cope with waves, and the wind speed increased up to 45kn. After the first day, the crew was subjectively stressed. These harsh conditions induced major seasickness and vomiting in one crew member, with a total inability to work on deck. This subject started to recover only at the end of the navigation, during which he never ate and vomited many times without being able to drink and rehydrate. Two other people vomited but were not impaired at work. Liquid reintegration started the day after. On the second day, the conditions changed, with waves height reduced to 1-2m SWH. The boat headed downwind, hoisting a code zero sail, maintaining the boat flat at an average speed of 9-10kn with 15-25kn of wind speed. The navigation remained stable until the end of the navigation in Lisbon on the third day.
Ethical considerations
The study was conducted following the Helsinki Declaration and was approved by the Ethical Committee of the University of Milan, Italy (Aut. n° 37/17). All the volunteers signed written informed consent.
Motion Sickness Questionnaire
To study motion sickness, previously validated Global Sickness Rating Scale (GSRS) (Golding et al. 2003) and Motion Sickness Questionnaire (MSQ) (Golding 2016) were used.
Saliva and Urine collection
Approximately 1 mL of saliva was obtained before and after the training and collected in Salivette devices (Sarstedt, Nümbrecht, Germany) at 8 AM. The subjects were trained on the correct use as previously reported (Mrakic-Sposta et al. 2019; Mrakic-Sposta et al. 2020).
Urine samples were collected by voluntary voiding in a sterile container before and after the training and every day during the training and navigation at 9 AM, 3 PM, and 9 PM according to the 3 h shifts. All samples were stored at 4°C in a portable cooler on board and during the transport back to the laboratory. The specimens were then stored in multiple aliquots at -20°C until assayed and thawed only once before analysis.
ROS and TAC
An X-band Electron Paramagnetic Resonance spectroscopy (9.3GHz) (E-Scan, Bruker Co., MA, USA) was used to detect ROS production and TAC values. Saliva samples were stabilized at 37°C using a Temperature and Gas Controller ‘‘Bio III’’ unit (Noxigen Science Transfer & Diagnostics GmbH, Germany), interfaced with the E-Scan. ROS production and TAC assessment methods were previously described (Mrakic-Sposta et al. 2019; 2012). Samples were analyzed in triplicate.
Cortisol
The concentration of free cortisol in the saliva was quantitatively determined through ELISA method according to the manufacturer’s protocol (COR(Cortisol) ELISA Kit; FineTest, Wuhan Fine Biotech Co.) as previously described (Dorn et al. 2007).
8-isoprostane
Lipid peroxidation was assessed in urine by competitive immunoassay measuring 8-isoprostane concentration (8-iso-PGF2a) (Cayman Chemical, USA). The method was previously described (Bosco et al. 2018).
NO metabolites
NOx (NO2+NO3) levels were assessed in urine by a method based on the Griess reaction (Green et al. 1982), using a commercial kit (Cayman, BertinPharma, Montigny le Bretonneux, France). Methods were previously described (Mrakic-Sposta et al. 2019)(Green et al. 1982).
Every assessment was carried out in duplicate and read by a microplate reader spectrophotometer (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland).
Creatinine, Neopterin, and Uric Acid
Urinary creatinine, neopterin, and uric acid concentrations were measured by isocratic high-pressure liquid chromatography. The calibration curves were linear over the range of 0.125-1 mmol/L, 0.625-20 mmol/L, and 1.25-10 mmol/L for neopterin, uric acid, and creatinine levels, respectively. Inter-assay and intra-assay coefficients of variation were <5%. Methods were previously described (Mrakic-Sposta et al. 2019; Glantzounis et al. 2005).
Urine standard analysis
The Urine Test Strips (Combi screen 11sys PLUS, GIMA, Gessate, Milan, Italy) were used to semi-quantitative determinations of bilirubin, urobilinogen, ketones, proteins, blood, pH, leukocytes, and specific gravity/density in urine. The tests were performed in duplicate.
Statistical analysis
Statistical analysis was performed using the GraphPad Prism package (GraphPad Prism 9.0.1, GraphPad So ware Inc., San Diego, CA). Data are presented as mean ± SD. Statistical analyses were performed using: non-parametric tests, Wilcoxon matched-pairs signed-rank test for independent samples (ROS and TAC in saliva), due to the small sample size for compared pre vs. post and ANOVA repeated measures, with multiple comparison tests to further check the among-groups significance. p<0.05 was considered statistically significant. Change Δ% estimation [((post value-pre value)/pre-value)*100] is also reported in the text. Non-parametric Spearman correlation (r) with 95% confidence intervals was used to detect possible relationships between selected parameters.