Oxidative Stress and Motion Sickness during Offshore Sailing

Purpose: Offshore Ocean Sailing is a highly demanding activity in which subjects are exposed to psychophysical stressors for a long time. To better dene the physiological adaptations, we investigated the stress response of subjects exposed to 3-days long ocean navigation with disruption of circadian rhythms. Methods: 6 male subjects were involved in the study and provided urine and saliva samples before setting sail, during a single day of inshore sailing, during 3-days long ocean navigation, and at the arrival, to measure oxidative stress, cortisol, nitric oxide metabolites (NOx) and metabolic response. Motion Sickness questionnaires were also administered. Results: The crew suffered a mean weight loss of 1.58 Kg. After the long navigation, a signicant increase in ROS production and decreased antioxidant capacity and uric acid levels were observed. Lipid peroxidation, NOx, ketones, creatinine, and neopterin levels were also increased. A signicant increase in cortisol levels was recorded, but no correlation was found with motion sickness questionnaires that were instead correlated with the rise of NOx. Conclusions: physical and psychological stress response derived from offshore sailing resulted in increased oxidative stress, nitric oxide metabolites, and cortisol levels, unbalanced redox status, transient renal function impairment, and ketosis. A direct correlation between motion sickness symptoms evaluated through questionnaires and NOx levels was also found.


Introduction
Sailing is a worldwide popular activity that includes various types of boats and disciplines. Offshore Ocean Sailing (OOS) is considered one of the most extreme endurance sports, exposing the crew to longlasting, physically and psychologically demanding efforts (Allen and De Jong 2006). OOS usually implies prolonged periods -ranging from days to months -spent at sea, in an extremely harsh environment, in isolation and self-su ciency, far from safe harbors and with limited access to external aid or rescue (Sjøgaard, Inglés, and Narici 2015). The boat represents an extremely uncomfortable, cold, wet, unstable, and enclosed environment without any privacy or comfort. In particular, racing boats are performanceoriented and with little comfort onboard. Energy expenditure during offshore sailing is high (Myers et al. 2008), and an adequate nutrient intake is challenging to maintain onboard, especially during harsh weather conditions (Fearnley et al. 2012). Negative energy balance often results in weight loss, decreased body fat percentage, and reduced muscle strength, proportionally to the length of the race (Bigard et al. 1998; Lafère et al. 2020). Proper sleep management is also essential to maintain adequate performance (Léger et al. 2008). Sailors adopt polyphasic sleep techniques and incur severe sleep restrictions during competition, thus resulting in cognitive performance and alertness decrease (Hurdiel et al. 2014).
The study of adaptations to extreme environments is gaining popularity. Nonetheless, the literature exploring short-and medium-term adaptations to OOS is still insu cient. Seafarers are also exposed to high and prolonged stress levels. Loneliness, circadian rhythms disruption, and fatigue often result in alterations in their physical (Oldenburg et al. 2013) and psychological domains (Carotenuto et al. 2012).
Due to the constant instability, the maritime environment signi cantly impacts cognitive and neuromuscular activity (Pisula, Lewis, and Bridger 2012).
Moreover, motion sickness often affects people exposed to transportation and visual instability through a sensory mismatch mechanism between perceived and expected stimuli. A large percentage of people experience seasickness, with higher work-related risks and detrimental effects on the performance of sailors and seafarers (Golding 2016;). Consistently, high-stress levels can induce a modi cation in normal circadian uctuations of cortisol, a glucocorticoid whose peak level in normal conditions is recorded after awakening (Stalder et al. 2016). During OOS and other highly stressing activities, a attening of this curve has been recorded, with sustained high cortisol levels throughout the effort (Gunnarsson et al. 2004).
During inshore and offshore sailing, physical effort is inconstant, characterized by high intensity and anaerobic bursts, with increases in oxygen consumption and heart rate ( This study aimed to investigate oxidative stress variations in sailors involved in OOS. To have a more accurate de nition of stress, we also evaluated cortisol levels, biochemical pro le, and renal function markers creatinine and neopterin. Motion Sickness has been investigated through neurophysiological symptoms questionnaires.

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 ve 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 rst day and night, the atmospheric conditions were challenging. Swell of 3/3.5 meters Signi cant 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 signi cant squalls and had to ee 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 rst 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 at 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 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).

Urine standard analysis
The Urine Test Strips (Combi screen 11sys PLUS, GIMA, Gessate, Milan, Italy) were used to semiquantitative determinations of bilirubin, urobilinogen, ketones, proteins, blood, pH, leukocytes, and speci c 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 signi cance. p<0.05 was considered statistically signi cant. Change Δ% estimation [((post value-pre value)/pre-value)*100] is also reported in the text. Non-parametric Spearman correlation (r) with 95% con dence intervals was used to detect possible relationships between selected parameters.

Results
A signi cant difference (p < 0.05) was observed in Weight (Kg) between Pre and Post ( Table 1). All crew members suffered a loss of weight (Mean weight loss: 1.58kg).  Fig. 2H).
No signi cant differences were recorded in GSRS for different items and MSQ during the three days of offshore navigation (see Tables 2 and 3).  Urine standard parameters are reported in Table 4. A signi cant increase in urinary ketones levels was detected during the navigation. pH and bilirubin values also increased but did not reach statistical signi cance.   (Fig. 3D), and stomach awareness r = 0.98 (p = 0.0007) (Fig. 3E); during the 3rd day no correlation was found.

Discussion
To our knowledge, this is the rst study to investigate oxidative stress on urine and saliva sampled from non-professional sailors during OOS and possible correlation with motion sickness. This setting is particularly challenging, with rapid changes in terms of environmental conditions and circadian rhythms.
According to the results, subjects exposed to OOS suffer a signi cant multifactorial increase in oxidative stress and cortisol. A small number of studies considered modi cations in cortisol levels in sailors and seafarers. Oldenburg et al. found that cortisol awakening levels were highly dependent on subjective stress perception and work type. Mental work was also associated with higher cortisol levels than physical work (Oldenburg and Jensen 2019). This is con rmed by cortisol levels found in maritime pilots, increasing their tasks' di culty (Main et al. 2017). Some studies' results re ect that seafarers' cortisol levels are higher in port stays than at sea, probably because of the break of a working routine found during days at sea ( . Nausea caused by motion sickness is also characterized by gastric dysrhythmias (Koch 2014). In accordance with these studies, we found a signi cant linear relationship between NOx levels and GSRS during the rst day of navigation, during which the subjects suffered the most intense motion sickness (Fig. 3).
During inshore regattas, short bursts of high-intensity activity are described . In America's Cup sailors, oxidative stress markers after the race were higher than their baseline levels, especially in crew members involved in high-intensity physical work (Barrios et al. 2011). Our study is the rst to analyze oxidative stress markers during OOS.
Our results show a signi cant increase in ROS production after the navigation. The imbalance between the ROS production rate (about + 100%) and the antioxidant scavenging (− 12%, see Fig. 2A, In our study, an increase of evaluated biomarkers concentrations was observed during and post-offshore sailing and was associated with ROS production. In any case, this study did not assess the chronic or long-term effects of offshore sailing. Mainly referred to kidney activity, the subjects manifested a temporary "impairment of renal function" as a likely physiological or adaptive response to dehydration. This could also be linked to signi cant weight loss (see Table 1) and vomiting, which changes ketones concentration, pH, and speci c density (see Table 4). Ketones increase could also hint at how athletes' metabolism copes with high energy demand and stress. Their production is stimulated by low insulin, high glucagon, and epinephrine concentrations, suggesting a shift to metabolic e ciency and fuel sparing of the organism exposed to endurance exercise and fasting (Cox et  Considering that sailors are exposed to harsh environmental conditions and that motion sickness and working rates can in uence nutrition habits during a race (Fearnley et al. 2012), it is of utmost importance to maintain an adequate water intake during navigation to prevent renal damage and to keep proper caloric intake to sustain physical performance.

Limitations And Conclusions
As for other studies (Galvani et al. 2015;Gunnarsson et al. 2004;Fearnley et al. 2012;Lafère et al. 2020) that focus on OOS, we found many di culties in producing reliable data and scienti c evidence. The researcher himself, which was part of the crew, had to take part in the strenuous activity schedule, the space for medical devices and samples on board is limited, invasive procedures are complicated to perform because of continuous motion, electronic devices cannot be charged because electrical power is limited and needed for navigation. The complexity of this environment often results in a lack of reliable literature (Allen and De Jong 2006). Therefore, we chose to obtain urine and saliva samples because of the limited logistic disadvantages of these samples.
A limitation of this study is the lack of data on the quality and duration of sleep. This might have in uenced the cortisol level, but Liberzon  Another limitation is that we have not been able to obtain information on sailors' cardiovascular and metabolic activity during the navigation, even though they have been described in other similar and comparable studies (Galvani et al. 2015;Neville et al. 2009). In the future, we hope we will be able to implement our methods and obtain this data in a similar environment.
However, the present offshore sailing study offers valuable information on the redox state, renal function, and motion sickness response during this high demanding activity. OOS has been shown to induce an increase in oxidative stress biomarkers and NO metabolites. A correlation was found also between the increase in NO metabolites level and motion sickness intensity evaluated through questionnaires and symptoms. In this experiment, a transient reduction in renal function was found. Moreover, salivary cortisol increased in response to physical activity and stress induced by navigation. Future studies are required to investigate the biochemical processes and the clinical correlations consequent to maritime exposure.  ). TAG contributed to the study design, data collection and drafting of the manuscript; AV contributed to the data analysis, interpretation and critical review of the manuscript; CD contributed to data analysis. MP and DC contributed to the study design, and critical review of the manuscript. GB contributed to the study design, data interpretation, and critical review of the manuscript. SMS contributed to the study design, data analysis, interpretation and drafting of the manuscript. GB and SMS con rm that the study objectives and procedures are honestly disclosed. All the authors approved the nal version of the manuscript

Funding
No funding was received for conducting this study.
Data availability the datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.

Con ict of interest
The Authors have no relevant nancial or non-nancial interests to disclose.

Ethics approval
This research study was approved by the Ethical Committee of University of Milan, Italy (Aut. n° 37/17).
All procedures conformed to the standards set by the 1964 Declaration of Helsinki and its later amendments.

Consent to participate
Informed consent was obtained from all individual participants included in the study Consent for publication all authors have read the manuscript and expressed their consent for the publication