Physiological stress responses of European sea bass Dicentrarchus labrax related to changing operations of floating net cages in intensive farming conditions at the Moroccan M'diq Bay
Abstract
The present study aimed to quantify and compare in situ the primary and secondary physiological stress responses, related to the changing operations of floating net cages, in both subadult (523 days post hatching [dph]) and adult (916 dph) European sea bass Dicentrarchus labrax under intensive farming conditions in the Moroccan M'diq Bay. The blood levels of cortisol, glucose, total cholesterol, total protein, and lactate, as well as the percentage of haematocrit, were measured before and after this operation. The results showed significantly elevated levels of cortisol and blood glucose in both age groups, whereas total cholesterol and protein levels were unaffected. In fact, blood lactate significantly decreased in subadults, whereas in adults this parameter was not affected by the operation. However, the haematocrit percentages measured after the operation were significantly higher than those found before the operation in both groups of fish, which is attributed to the increased rate of oxygen renewal in the new net cages and the lower water temperature inside the cages. With regard to the age-specific response during this essential operation plasma cortisol, blood glucose, and lactate concentrations, as well as plasma total protein levels, were significantly higher in subadults than in adults, in both pre- and post- stress measurement, with the presence of individual-specific response. It is concluded that aquaculture practices such as changing the aquaculture net cage could have repercussions in terms of the classic physiological responses to stress in D. labrax.
1 INTRODUCTION
The European sea bass, Dicentrarchus labrax, not only is known for its nutritional and health benefits (Periago et al., 2005) but also holds great commercial and aquaculture significance, particularly in the Mediterranean coastal regions. Indeed, the industrial aquaculture of this species in Morocco is at the embryonic stage; only three operational marine fish farms, specialized only in the pre-grow out and grow out of this fish, have been established in the Mediterranean (M'diq Bay and Nador Lagoon) and Atlantic (Dakhla Bay) facades. However, Moroccan aquaculturists aim to ensure the sustainability of this sector by the managing environmental issues and the maintaining fish welfare (Bahida et al., 2022; Cheyadmi et al., 2023; Nhhala et al., 2022).
During sea bass farming, fish are exposed to various external (environmental) and internal (biological) stress factors, which can negatively impact their welfare and health. Environmental stressors include sudden changes in seawater biotic conditions (Cámara-Ruiz et al., 2021; Rosado et al., 2022), as well as abiotic conditions (Alfonso et al., 2020; Barboza et al., 2018; Debusschere et al., 2016; Liu et al., 2022; Masroor et al., 2018). Additionally, the daily or periodic aquaculture procedures can contribute to stress levels in the fish (Cheyadmi et al., 2023; Fanouraki et al., 2011; Ferrari et al., 2020; Poltronieri et al., 2007; Rubio et al., 2010; Sadoul et al., 2021; Samaras et al., 2018). Moreover, biological factors such as variation in fish species, variation in genetic background, age and size, and individual coping styles of D. labrax, in addition to the lifetime history of stress exposure (Alfonso et al., 2023; Cadiz et al., 2018; Cheyadmi et al., 2023; Fanouraki et al., 2011; Ferrari et al., 2020; Vandeputte et al., 2016), could also interact with the environmental stressors-related aquaculture on the various stress responses of D. labrax, which vary within and between fish species.
Acute or chronic confinement, in high-rearing densities, increases blood cortisolemia, glycaemia, lactatemia, and osmolarity (Samaras et al., 2021; Vazzana et al., 2002), as well as the levels of non-esterified fatty acids (Di Marco et al., 2008). These physiological responses were found to be associated with a decrease in oxygen consumption and respiratory capacity of fish (Lupatsch et al., 2010). They were also linked to reduced feed consumption and body weight (Samaras et al., 2018). The manipulation and transfer of fish using dip-nets in a new experimental tank can result in various physiological responses and stress-related effects. These include upregulation of heat-shock protein 70 genes, increased presence of rodlet cells in the intestinal lumen and kidney of stressed fish, and elevated levels of oxidative stress immunoprecipitated such as heat-shock proteins, malondialdehyde, nitrotyrosine, and 4-hydroxy-2-nonenal in different organs (Fiocchi et al., 2020). These effects are often accompanied by an increased rate of ammonia nitrogen excretion after the operation (Kayali et al., 2011).
Sampling operations involving regular height and weight measurements have been found to induce an increase in blood cortisolemia, glycemia, and lactatemia. These physiological changes are correlated with a decrease in proteinemia levels and haematocrit percentage. Additionally, fish subjected to these sampling operations exhibit active swimming behavior and intergroup interactions. Furthermore, there is an increase in the mortality rate after the operation (Cheyadmi et al., 2023).
For the stress responses related to fish age and/or size, adults were more sensitive to stressors than small fish (Alfonso et al., 2023; Cheyadmi et al., 2023; Fatira et al., 2014; Poltronieri et al., 2007). However, the situation was complex in this sentient fish; the stress responses could be varied between the same age exposed to various stress types, based on the intensity, the time course, and the repeatability of stressors and its predictability by individual fish.
Changing the net in the cages is an essential and regular operation during the reproductive cycle of sea bass, when the fish grow, thus, when the colonization of the fouling prevents the circulation of sea water in the cage. This aquaculture operation mainly affects the welfare of the fish and their growth. There are currently no published quantitative data on the in situ (offshore) physiological stress effects of the steps of this aquaculture operation in farmed European sea bass, as well as in other fish species.
Here, we aimed exclusively to quantify the stress response of D. labrax related to this operation by analyzing the primary and secondary physiological stress indicators. The measurement of the following blood indicators is useful to show their impacts on the welfare of this fish. Cortisol is the main stress hormone in fish. Glucose and lactate (carbohydrates), total cholesterol (lipids), and total proteins represent the major aerobic and anaerobic energy metabolites in these aquatic animals. In this context, haematocrit percentages indicate their health status.
2 MATERIALS AND METHODS
2.1 Ethics statement
This study has been approved by the guidelines of the Council of the European Union (2010/63EU), and of the Fisheries Society of the British Isles, dedicated to the protection of freedoms of fish welfare during handling and scientific experiments in the aquaculture farm and laboratory (FSBI 2002; UE Directive 2017).
2.2 Biological material and in situ location
Two age groups of sea bass D. labrax, under commercial culture conditions, in floating cages at Aqua M'diq fish farm were studied. Twenty subadult fish (523 days post hatching [dph]) and 20 adult fish (916 dph) were randomly fished using a landing net before and after changing the aquaculture net cages (10 fish per state of stress). The morphometric parameters of each fish were measured using an ichthyometer for body length and a precision scale (0.01 g) for body weight.
Aqua M'diq fish farm is located in the Moroccan Mediterranean east of the M'diq Bay (35° 41′ 16.3″ N, 5° 19′ 23.1″ W, between Sebta Cap in the north and Negro Cap in the south). The ecological limits, for the survival of D. labrax fish throughout the year in this farming environment, are characterized by a salinity of the order of 36.2 ± 1‰, temperature varies from 15 to 21°C, the concentration of dissolved oxygen varies from 7 to 8 mg/L, and the pH is 8, and the concentration of varies from 0.002 and 0.05 mg/L (Cheyadmi et al., 2023; Nhhala et al., 2022).
2.3 Physical–chemical parameters of water
The physicochemical parameters of sea water—temperature, pH, and percentage of dissolved oxygen saturation—were measured, inside and around the cages, before and after changing the net of the floating cages, using a multiparameter probe HANNA (mode HI98196; from Morocco).
2.4 Aquaculture operation: changing the floating net cages
During the present study, D. labrax fish were made to fast the day before this operation (24 h). The sailors (n ⩾ 5) on the surface of the cages and the divers below (n = 2–3) reduced the rearing volume, using a rope tied in the center of the lower part of the net, and the weights of the net were untied and tied with temporary ropes to the pipes of cage handrails. They installed the new net cage below the old net, tying it well to the cage raft frame and tubes, then they lifted the old net cage to loosen the fish and allow them to swim into the clean one, taking care that the fish are not trapped in the folds of the old net. In the end, they put the weights of the new net back below and ensured that the operation goes well, the net was fixed well so that the fish do not escape. The sailors then distributed the feed to the fish, and the divers controlled the swimming and feeding behaviors of the fish. This operation lasted c. 40 min to 1 h; it requires the strength and collaboration of several sailors and divers because of the heavy mass of the net installed and that need to be removed manually, in addition to the current and tide conditions.
- Pre-stress state: immediately after the reduction in the rearing volume (10 fish per age group).
- Post-stress state: after the installation of the new net cage and the elimination of the old one (10 fish per age group).
2.5 Blood collection and stress-biomarker analysis
The fish were anaesthetized with clove oil immediately after fishing, they were then placed in ice to prevent them from drying out and facilitate blood sampling. Then their tail vein was punctured (21G needle precision; 2.5-mL syringe capacity). The blood samples were collected on the speedboat at M'diq offshore. The blood was stored in heparinized tubes (4 mL) in a cooler at 4°C.
The procedure followed for the quantification of physiological stress response induced by the present operation was the same established in our recent study (Cheyadmi et al., 2023). For each fish, blood glucose and total cholesterol levels were measured using a portable reader and specific strips (BeneCHEK TM Plus Multi-Monitoring System, USA), blood lactate level was also measured using a lactatometer and specific strips (THE EDGE Blood Lactate monitoring system, China), and plasma proteinemia was measured using a portable optical refractometer (Brix at 20°C, BK-PR32, China) after centrifuging blood (1250g for 15 min, Sigma Centrifuge). The percentage of blood haematocrits was measured as follows: (volume of red blood cells/total volume of blood) × 100. The plasma obtained was collected in Eppendorf tubes and stored at −20°C for the quantitative determination of plasma cortisol concentrations (Fish Cortisol ELISA Kit–Competitive, Cusabio Biotech Co., Ltd., Germany) at the ONSSA laboratory in Tangier. Optical densities were measured at 450 nm for at least 5 min, using a microplate spectrophotometer (Biotek-Elx 808, USA) connected to a computer, and they were processed using Gen5 and Curve Expert 1.4 software, to calculate the concentrations of the standards and the fish plasma samples.
2.6 Statistical analysis
The data obtained during the present study were statistically analysed using IBM SPSS Statistic (version 25) with a significance risk α = 0.05. The values of the morphometric parameters and the physiological indicators of stress presented in the text and the tables were expressed as mean ± standard deviation (minimum–maximum).
The distribution and comparison of physiological stress responses (for plasma cortisol, blood glucose, lactate, total cholesterol, plasma total protein levels, and haematocrit percentage), induced by the aquaculture operation “changing floating net cages,” in both fish groups in pre- and post-stress states, were statistically analysed using the Shapiro–Wilk normality test (n = 10 < 30), the non-parametric test (Mann–Whitney U-test), bidirectional multivariate analysis of variance (ANOVA) (two-way ANOVA) via Levene's test (homogeneity of variance), Student's t-test (comparison of means), and post hoc correction of Bonferroni with between-subjects effects test.
3 RESULTS
3.1 Presentation of age and morphometric parameters
Table 1 presents the results of biometric and morphometric parameters of 20 subadult and adult European sea bass. The average weight and size of subadult fish were 154.55 ± 28.05 g and 23.07 ± 1.14 cm, respectively, and those of adult fish were 494.25 ± 54.62 g and 35.26 ± 1.39 cm, respectively.
| European sea bass | Age | Days of stay at the farm | Weight (g) | Length (cm) |
|---|---|---|---|---|
| Subadults (N = 20) | 523 dph | 404 days (⩾ 1 year) | 154.55 ± 28.05 (130–235) | 23.07 ± 1.14 (21.3–25) |
| Adults (N = 20) | 916 dph | 698 days (≈2 years) | 494.25 ± 54.62*** (440–600) | 35.26 ± 1.39*** (33–38) |
- Note: Values are mean ± standard deviation (minimum–maximum), with significance codes; ***p < 0.001; two-way ANOVA.
- Abbreviation: dph, days post hatching.
3.2 Variation in the physicochemical parameters of the water in the offshore breeding cages before and after changing the farming nets
Before the changing operations of floating net cages, the mean percentage of dissolved oxygen saturation (% O2) in the farm seawater was 90%. However, the mean percentage found inside the cages housing the D. labrax subadults and adults was around 70%. Statistically, the percentages of dissolved oxygen in the cages housing the D. labrax subadults and adults were significantly lower than those measured in the seawater of the farm (p < 0.001 for the two cages). After the nets of the two rearing cages were changed, the percentage of dissolved oxygen saturation increased significantly (p < 0.01), and the values of temperature and pH decreased in the sea water inside the two cages compared to those recorded before changing the nets (Table 2).
| Sea bass subadults | Sea bass adults | |
|---|---|---|
| Outside the cage | T = 19.46 ± 0.03°C % O₂ = 88 ± 2% pH = 8.41 ± 0.02 |
T = 19.51 ± 0.03°C % O₂ = 91 ± 1% pH = 8.43 ± 0.01 |
| Inside the cage before changing the aquaculture net | T = 19.65 ± 0.05°C** % O₂ = 71 ± 2%*** pH = 8.45 ± 0.03NS |
T = 19.55 ± 0.01°CNS % O₂ = 73 ± 2%*** pH = 8.48 ± 0.01** |
| Inside the cage after changing the aquaculture net | T = 19.53 ± 0.06°CNS % O₂ = 83 ± 1*** pH = 8.45 ± 0.02NS |
T = 19.48 ± 0.10°C* % O₂ = 81 ± 1%** pH = 8.46 ± 0.01NS |
- Note: Two-way analysis of variance to compare the parameters of the sea with those in the cage before the operation and in the cage before and after the operation. Values are mean ± standard deviation, with the significance thresholds.
- NS p > 0.05.
- * p < 0.05.
- ** p < 0.01.
- *** p < 0.001.
3.3 Physiological responses of subadult and adult sea bass during the operation of changing the net cages
The Shapiro–Wilk test (n = 10 < 30), the Q–Q plot, and Henry's line showed the normality of the mean concentrations recorded for all the biomarkers measured in the two stages of the sea bass in two states pre- and post-operation as p-values > 0.05.The pre-stress values of blood biomarkers, found in subadults and adults D. labrax, were as follows: 157.19 ± 71.51 ng/mL and 136.92 ± 31.79 ng/mL for cortisolemia, 12.34 ± 5.09 mmol/L and 7.11 ± 2.98 mmol/L for glycemia, 15.12 ± 1.92 mmol/L and 3.83 ± 2.13 mmol/L for lactatemia, 5.60 ± 1.05 mmol/L and 6.18 ± 1.97 mmol/L for cholesterolemia, 7.34 ± 1.07 g/dL and 5.95 ± 0.86 g/dL for proteinemia, and 46.87 ± 9.60 % and 47.70 ± 7.86 % for the hematocrit percentage, respectively. However, the post-stress values recorded were: 290.37 ± 113.23 ng/mL and 207.58 ± 62.65 ng/mL for cortisolemia, 16.75 ± 3.63 mmol/L and 11.74 ± 4.68 mmol/L for glycemia, 8.72 ± 3.77 mmol/L and 3.71 ± 2.31 mmol/L for lactatemia, 6.15 ± 1.10 mmol/L and 6.37 ± 0.84 mmol/L for cholesterolemia, 7.20 ± 1.25 g/dL and 6.74 ± 0.85 g/dL for proteinemia, and 53.12 ± 8.05 % and 54.62 ± 9.88 % for the hematocrit percentage, in D. labrax subadults and adults, respectively (Table 3).
Generally, the between-subjects effects test (two-way ANOVA) showed that the categories “age” (F = 21.692, ddl = 6, and p < 0.001), “state of stress” (F = 11.023, ddl = 6, and p < 0.001), and interaction of “age and state of stress” (F-value = 4.587, ddl = 6, and p = 0.002) will have a statistically highly significant effect on the distribution of blood biomarkers in sea bass during the net-cage changing operation. For subadult sea bass, the “stress state” category will have a very highly statistically significant effect (F = 14.263, ddl = 6, and p < 0.001), on the recorded concentrations of blood indicators, during the present study, unlike for adult sea bass (F = 2.427, ddl = 6, and p = 0.085).
Statistical tests, Mann–Whitney U-test, two-way ANOVA, Levene's and Student's t-tests, and Bonferroni post hoc correction, showed a significant increase in mean values of cortisol levels (p < 0.01) and blood glucose levels (p < 0.05), related to the operation, in both subadult and adult sea bass. In addition, whereas subadult sea bass showed a very highly significant decrease in mean blood lactate levels (p < 0.001), adults did not show any significant variation between pre- and post-operation values in blood lactate levels. In post-operation, the levels of plasmatic proteins of subadults did not show any difference, whereas they were slightly increased in adult fish, but this change was not statistically significant in terms of “stress state” category (p > 0.05), as presented in Table 3.
| Floating net cages changing operation | |||||||
|---|---|---|---|---|---|---|---|
| European sea bass | Subadult fish (N = 20) | Adults fish (N = 20) | p-Value | ||||
| Stress indicators | Pre-stress (N = 10) | Post-stress (N = 10) | Pre-stress (N = 10) | Post-stress (N = 10) | Age | Stress state | Age and stress state |
| Cortisolemia (ng/mL) | 157.19 ± 71.51 (58.90–290.61) | 290.37 ± 113.23** (147.80–512.80) | 136.92 ± 31.79 (88.27–147.06) | 207.58 ± 62.65** (119.27–311.70) | 0.038 | 0.000 | 0.199 |
| Glycemia (mmol/L) | 12.34 ± 5.09 (5.72–20.09) | 16.75 ± 3.63* (11.21–22.81) | 7.11 ± 2.98 (2.83–11.77) | 11.74 ± 4.68* (5.66–19.76) | 0.000 | 0.002 | 0.937 |
| Lactatemia (mmol/L) | 15.12 ± 1.92 (11.77–18.32) | 8.72 ± 3.77*** (1.33–13.32) | 3.83 ± 2.13 (1.67–8.21) | 3.71 ± 2.31 (1.11–7.99) | 0.000 | 0.000 | 0.001 |
| Cholesterol (mmol/L) | 5.60 ± 1.05 (3.57–6.78) | 6.15 ± 1.10 (4.17–7.30) | 6.18 ± 1.97 (3.60–10.25) | 6.37 ± 0.84 (5.18–7.87) | 0.345 | 0.384 | 0.667 |
| Total protein (g/dL) | 7.34 ± 1.07 (5.88–9.00) | 7.20 ± 1.25 (5.10–8.40) | 5.95 ± 0.86 (4.74–7.14) | 6.74 ± 0.85 (5.34–7.80) | 0.007 | 0.323 | 0.161 |
| Haematocrit (%) | 46.87 ± 9.60 (33.33–60.00) | 53.12 ± 8.05 (35.00–62.50) | 47.70 ± 7.86 (36.00–58.82) | 54.62 ± 9.88 (39.00–70.00) | 0.683 | 0.025 | 0.909 |
- Note: Values are mean ± standard deviation (minimum–maximum). The significance codes are shown using two-way analysis of variance (Levene's and Student's t-tests and Bonferroni's post hoc correction) and Mann–Whitney U-test.
- * p < 0.05.
- ** p < 0.01.
- *** p < 0.001.
Furthermore, the average concentrations of cortisol, glucose, lactate, and total proteins measured in subadults, before and after the operation, were statistically very high compared to those measured in adults, with p < 0.05, p < 0.001, p < 0.001, and p < 0.01, respectively (between-subjects effect—two-way ANOVA). These results indicate the presence of stress responses related to the age of D. labrax. Levene's test showed a significant wide dispersion of blood cortisol, glucose, and lactate concentrations, measured pre- and post-operation, in both age groups. These results indicate the presence of stress responses related to the individual status of D. labrax.
Our results showed a slight increase in the average total cholesterol levels in both age groups post-operation, but the recorded levels showed no statistically significant variation based on age difference (p > 0.05) and state of stress quantification (p > 0.05).
In addition, the present results revealed a statistically non-significant increase (p > 0.05) in the blood level of the percentage of haematocrits for the two life stages, subadults and adults, after the operation. Indeed, the between-subjects effects test showed, in general, the effect of the “state of stress” category on the percentage of haematocrits after the operation with p < 0.05.
4 DISCUSSION
The major concerns of Moroccan aquaculturists are accentuated on the development and sustainability of sea bass farming and the opening up to other marine fish (gilthead seabream Sparus aurata and common meager Argyrosomus regius), using good aquaculture practices and the prevention and management of environmental issues and stress episode effects on aquatic animals' health and welfare, to minimize their damage, including the interest of this present study. Here, we focused on in situ (offshore) quantification and comparison of the primary and secondary physiological stress responses due to the aquaculture operation of changing cage nets, in both subadult (523 dph) and adult (916 dph) sea bass D. labrax, farmed in the Mediterranean M'diq Bay, Moroccan coast.
Our results showed the statistically significant increase in terms of the nonspecific stress biomarkers (Dinardo et al., 2020), in both age groups of sea bass, mainly hypercortisolemia (p < 0.01) associated with hyperglycemia (p < 0.05), after the installation of the new net of the two cages housing these fish. However, adults showed significantly lower concentrations than subadults (p < 0.05 for plasma cortisol level and p < 0.001 for blood glucose level) during this operation. The literature reveals that the primary stress glucocorticoid “cortisol” modulates carbohydrate metabolism “blood glucose and hepatic glycogen” via the activation of gluconeogenesis in marine and freshwater fish, under various rearing conditions, allowing proper mobilization and distribution of energy resources to various fish tissues (Lawrence et al., 2019; Mommsen et al., 1999; Schreck & Tort, 2016). This hypercortisolemia and then hyperglycemia, appearing during the changing of the net cages, are often immediate and transient mediators of the primary and secondary stress responses, respectively (Schreck & Tort, 2016; Vazzana et al., 2002), which could be due to the presence of the sailors on the surface and the divers below, to the surrounding voices, and to the operation steps (i.e., the decrease in the breeding volume to facilitate the installation of the new net on the old net, to shelter the fish well, and the pulling of the old net gently, to release fishes toward the new net). We could also explain the increase in the concentrations of these two biomarkers during this aquaculture operation as an adaptation pathway (Goikoetxea et al., 2021) or a defense mechanism (Lupatsch et al., 2010), exercised by subadult and adult sea bass to cope with this event that requires significant energy, and which is reflected visually by the active change in their swimming behavior “active or abnormal swimming and continuous disruption of social interactions” in the cage. Our results agree with all the studies carried out on the European sea bass, showing that their acute exposure to a stressful event—such as certain aquaculture activities and changes in farming environmental conditions—activates the hypothalamic–pituitary–interrenal (HPI) axis, represented by the increase in plasma cortisol levels associated with hypercirculation of blood glucose (Cheyadmi et al., 2023; Fanouraki et al., 2011; Lupatsch et al., 2010; Millot et al., 2014; Petochi et al., 2011; Santulli et al., 1999; Serradell et al., 2020; Vazzana et al., 2002; Yildiz & Altunay, 2011).
In industrial aquaculture, the low dissolved oxygen uptake by fish during their growth could be due to the small mesh width or clogging of the net cages, associated with a delay in aquaculturists changing the nets due to excessive bad weather or poor planning and management strategies. Moreover, these hypoxic conditions could result in detrimental damage to fish welfare, by stimulating physiological and hematological stress responses in fish and by affecting their metabolic and respiratory functions, their swimming and feeding behaviors, and their zootechnical performances (Alfonso et al., 2020; Cadiz et al., 2018; Simi et al., 2017; Vanderplancke et al., 2015). The physicochemical parameters of the breeding environment of sea bass, typically water temperature and dissolved oxygen rate, are the determining factors of fish welfare and health, as well as modulators of the respiratory and metabolic functions of fish. Our results showed that the percentages of dissolved oxygen, inside the cages housing both subadults and adults, pre-operation, are significantly lower than those measured in the farm sea water. And after the net cages were changed, the percentage of this parameter inside the two cages significantly increased (p < 0.001) and the temperature decreased. Claireaux and Lagardère (1999) showed that sea bass could maintain the normal rate of oxygen consumption during the first moments of their exposure to hypoxic conditions, before the conditions became more severe over time, associated with an increase in metabolic needs and therefore energy expenditure, as well as an increase in water temperature.
During the clogging of the farming net cage by fouling, the water renewal rate and dissolved oxygen saturation in the cage are limited, and therefore, the fish could be exposed to hypoxic conditions (Alfonso et al., 2020). The lactate level is relatively much higher in subadults (15.12 ± 1.92 mmol/L) before the operation, showing their reaction to avoid asphyxia and to maximize the dissolved oxygen uptake, via active mobility under anaerobic conditions of these lightweight fish, whether at the surface of the net cage or at the net wall. The results obtained by Islam, Kunzmann, Henjes, and Slater (2021a) showed that hyperlactatemia is an indicator of the activation of anaerobic metabolism in European sea bass, associated with the energy efforts deployed by this fish under stressful conditions. Based on our results and in situ observations, we could support the hypothesis of the plasticity or adaptability of European sea bass D. labrax to change both their physiological and behavioral indicators, simultaneously, under hypoxic conditions and environmental threats, to enhance oxygen consumption and to avoid asphyxia-related mortality (Alfonso et al., 2020; Debusschere et al., 2016; Lefrançois & Domenici, 2006; Millot et al., 2014). Indeed, our results show a very highly significant hypolactatemia (8.72 ± 3.77 mmol/L, p < 0.001) after changing the net in the subadult stage, which is associated with an increase in the percentage of dissolved oxygen saturation in the cage, 83% in post-net change condition versus 71% in pre-net change condition (p < 0.001). Conversely, blood lactate levels in adults, in both pre- and post-stress states, are significantly very low compared to subadults (p < 0.001). This decrease could be explained by the low swimming activity exerted by these large fish in the cage, as well as the metabolic depression resulting from the low rate of oxygen consumption (Debusschere et al., 2016) during the days before changing the net cages. Debusschere et al. (2016) also showed that this in situ secondary physiological response—lower whole-body lactate levels—in sea bass D. labrax exposed to stress is often a sign that the fish is following the freezing strategy, which is typical of anxiolytic behavior in fish. It can be concluded that the high concentrations of blood lactate in subadults are often an indicator of the anaerobic capacities exerted by the fish to react to stressful situations, whereas it is an indicator reflecting the aerobic limitations of adult fish (Sinha et al., 2015), whose concentrations are significantly very small than subadults. This response can also be explained by the power of subadult sea bass to recover their internal homeostasis compared to adults.
Studies carried out in this context have shown that plasma total proteins—important nonspecific immune response biomarkers—play multiple functional and protective roles in multiple fish, namely the adjustment of ionic balance and elimination of certain microorganisms and their effects on fish health (Dinardo et al., 2020; Shourbela et al., 2021). Therefore, the effect of stressful events on this indicator is manifested by plasma hypoproteinemia, resulting from their catabolism after the activation of the HPI axis or due to perepheral proteolysis, which disrupts their metabolic and immune functions and affects the growth and development of skeletal muscle of these organisms, in the long term (Alves et al., 2021; Cheyadmi et al., 2023; Islam et al., 2020a; Islam et al. 2020b). Our results show that the aquaculture net-changing operation does not affect the plasma concentrations of total proteins in the two life stages of D. labrax (p > 0.05, stress state category). However, the concentrations found in adults are statistically very low (p < 0.01) compared to those measured in subadults. Islam, Kunzmann, and Slater (2021b) showed that low plasma protein and blood lactate levels associated with high blood cortisol and glucose levels, such as the case of adults in the present study, indicate a disturbance in ionic balance and osmotic imbalance in sea bass, whose fish exerts extra energy and depletes its energy resources to establish itself.
Moreover, Levene's test showed a significant wide dispersion of blood cortisol, glucose, and lactate concentrations, measured in pre- and post-net-cage changing operation, which is consistent with our previous results for the case of sampling operation for size and weight measurement (Cheyadmi et al., 2023). This individual-specific physiological response indicates that each individual fish follows a specific strategy, different from the other, to deal with stressful events and restore its internal homeostasis, namely individual coping styles (Alfonso et al., 2020; Fanouraki et al., 2011; Ferrari et al., 2020), and which could be influenced mainly by their genetic, hormonal, and physiological cascades underlying the stress response (Goikoetxea et al., 2021). Our results also showed the presence of age-specific stress responses, for which the concentrations of plasma cortisol, blood glucose, and lactate and the level of plasma total proteins were significantly higher in D. labrax subadults compared to adults. Contrary to our recent study, these physiological responses in adult sea bass, to sampling operation for size and weight measurement, were higher than those in subadult sea bass, reared in the same farming conditions (Cheyadmi et al., 2023). We could explain this difference in terms of individual- and age-related responses in European sea bass, during the present study, as well as in the different studies in the literature, by the complexity of the system involved in the physiological response to stress in this sensitive fish.
The percentage of haematocrits, a simplified form of measuring the number of fish erythrocytes (Witeska et al., 2022), plays a primordial role in satisfying oxygen homeostasis and demand of aerobic metabolism in D. labrax, as well as an indicator of fish health status and antimicrobial defense (Cadiz et al., 2017). We could attribute the statistically significant increase in this hematological parameter in subadults and adults (p < 0.05) to the increase in the percentage of dissolved oxygen saturation of farm sea water (p < 0.001), inside the cages, after changing their nets. Previous studies carried out on the same fish species have shown that the increase in the haematocrit percentages and therefore of hemoglobin concentration is positively correlated with the increase in the number and volume of red blood cells in sea bass, which indicates the improvement in osmosis and the maintenance of normal serum concentrations of osmotic ions (Na+, Cl−, and K+) after the change of cage nets and vice versa (Islam, Slater, Thiele, & Kunzmann, 2021c; Montgomery et al., 2022). It could be added that the increase in the number of red blood cells in the bloodstream of fish attributed to the high percentage of haematocrits in both age groups is probably due to the overexpression of hemoglobin–oxygen binding genes at the level of head-kidney and spleen (Cadiz et al., 2017), and/or of the activation of the adrenergic response of the cardiovascular system of these fish, by stimulating the Na+/H+ β-adrenergic exchanger of sea bass erythrocytes and contracting their spleen (Montgomery et al., 2022), after the secretion of the cortisol hormone (Lawrence et al., 2019) after this aquaculture operation, to increase the branchial and cutaneous consumption of dissolved oxygen and to increase their contribution to the blood and therefore to the tissues.
5 CONCLUSIONS
Numerous studies have shown that aquaculture practices induce cyclic and repetitive physiological, metabolic, enzymatic, immune, and oxidative stress, as well as behavioral stress responses. The operation of changing cage nets is essential to guarantee the health and welfare of European sea bass in commercial farming. The practice of changing nets had a direct impact on cortisol and blood glucose secretion levels and on haematocrit percentages for both age groups. Other physiological indicators such as blood lactate and plasma total protein showed age-specific responses. It can be concluded that aquaculture practices such as changing cage nets trigger an acute physiological responses to stress in sea bass D. labrax.
AUTHOR CONTRIBUTIONS
Conceptualization: Soumaya Cheyadmi, Housni Chadli, Bouchra El Yamlahi, Hassan Nhhala, and Hicham Chairi; methodology: Soumaya Cheyadmi, Housni Chadli, Bouchra El Yamlahi, Mohammed El Maadoudi, and Ayoub Kounnoun; software: Soumaya Cheyadmi; validation: Soumaya Cheyadmi and Hicham Chairi; investigation: Soumaya Cheyadmi, Housni Chadli, and Bouchra El Yamlahi; writing–original draft: Soumaya Cheyadmi; writing—review and editing: Soumaya Cheyadmi, Francesco Cacciola and Hicham Chairi; supervision: Housni Chadli, Bouchra El Yamlahi, Mohammed El Maadoudi, Hassan Nhhala, Francesco Cacciola, and Hicham Chairi; funding acquisition: Francesco Cacciola. All authors have read and agreed to the final version of the manuscript.
ACKNOWLEDGMENTS
We thank the sailors and the divers of the Aqua M'diq aquaculture farm for their help, efforts, and patience during the study day. We also thank the staff of the Regional Laboratory for Analysis and Research (ONSSA, Tangier) and all people who have contributed from far or near to this modest study.
FUNDING INFORMATION
The authors declare that no funds, grants, or other support were received during this study.
CONFLICT OF INTEREST STATEMENT
We declare that we have no conflict of interest in this article.
Open Research
DATA AVAILABILITY STATEMENT
The data sets generated and analysed during the current study are available from the corresponding author on reasonable request.
