Intensive aquaculture production is accompanied by stressful situations, which may affect not only physiological responses and homeostasis, but also profitability, production, and sustainability of aquaculture industry worldwide (Hassan et al., 2021). Such stress response is characterized by hormonal, physiological, metabolic, molecular, and genetic changes, which provide additional energetic resources to face adverse situations (Barton and Iwama, 1991; Wendelaar Bonga, 1997). However, these adaptive mechanisms if not efficient, may lead to maladaptive responses such as low performance, physiological imbalance, and eventually death (Tort, 2011). The direct cause-effect relationship between stress and fish adaptive responses requires investigation, mainly of sub-clinical aspects affecting growth performance, health, disease resistance, and consequently economic loss (Sung et al., 2011; Dawood et al., 2020; Xavier et al., 2020).

Protein, one of the elements of appropriate nutrition, plays an important role on growth, stress response, and health (Oliva-Teles, 2012; Peres et al., 2022). Indeed, it has been observed that protein and amino acids (AA) deficiency reduces plasma AA (Li et al., 2007; Aragão et al., 2008), thus impairing the synthesis of specific proteins with crucial roles on cell signaling, immune and antioxidant responses, cell adhesion, and cell turnover (Buxbaum, 2007). An adequate dietary protein level may reduce oxidative stress (Pérez-Jiménez et al., 2009; Castro et al., 2012) and improve stress resistance and immune response (Habte-Tsion et al., 2017; Zuo et al., 2017). Moreover, the adjustment of dietary protein and AA may provide precursors to sustain fish homeostasis and welfare (Costas et al., 2008; Wu, 2013). Besides, under adverse conditions, voluntary feed intake may be impaired, and a protein/amino acid-fortified diet may be required to avoid deficiency signs.

Indeed, studies have shown that fortification of the diets above the requirement level, with specific amino acids such as arginine, methionine, tryptophan, and taurine may increase stress and disease resistance (Costas et al., 2011; Bañuelos-Vargas et al., 2014; Machado et al., 2015; Huang et al., 2021). For Nile tilapia, dietary protein levels may modulate stress response induced by high stock density (Abdel-Tawwab, 2012; Hooley et al., 2014). Despite its importance, available data on the effects of protein and AA in health and stress resistance remain scarce. Therefore, the current study investigated the interactive effects of dietary digestible protein (DP) level, below and above Nile tilapia protein requirements, and common stressors in intensive aquaculture, such as thermal, transport, and size sorting, on hematological and biochemical response of Nile tilapia.

This study was conducted in Botucatu, São Paulo State, Brazil (22°50'46.2" S and 48°25'51.94" W). Research on animals was conducted according to the institutional committee on animal use (protocol 163/2012-CEUA).

At the end of the 110-day feeding trial and after fasting for 12 h, and at the end of each stress challenge, eight fish/treatment (one fish/tank) were randomly collected, anaesthetized with benzocaine solution (67 mg L¹), and bled for evaluation of the hematological profile. Blood was collected from the caudal vein using a tuberculin syringe rinsed with anti-coagulant (3% EDTA, Vetec, Quimica Fina Ltda, Duque de Caxias, RJ, Brazil).

Red blood cell (RBC) and leukocyte counts were determined by dilution and enumeration using a hemocytometer. Leukocyte differentiation was performed using blood extension stained with May-Grunwald-Giemsa stain according to Jain (1986). Differential counting was performed under a microscope at 100X in immersion oil. To establish the percentage of each cellular component, 200 cells were counted on each extension. Hemoglobin (Hb) was determined by the cyanomethemoglobin colorimetric method using a commercial kit (Gold Analisa Diagnostica, Belo Horizonte, MG, Brazil) according to Collier (1944). Hematocrit (Htc) percentage was determined using the microhematocrit method described by Goldenfarb et al. (1971). Mean corpuscular volume [MCV = (Htc × 10)/RBC] and mean corpuscular hemoglobin concentration [MCHC = (Hb × 100)/Htc] were calculated according to Wintrobe (1934). Total plasma protein (TPP) level was measured using a manual Goldberg refractometer (Model SPR – N, Atago CO LTD, Japan) after the blood was centrifuged at 5000 rpm for 15 min.

For the biochemical analyses, blood was collected from the caudal vein, as aforementioned, using a tuberculin syringe with no anticoagulant. After that, blood samples were centrifuged at 5000 rpm for 20 min, and supernatant serum was collected. Albumin concentration (ALB) was determined by the bromocresol method using the commercial kit Albumina Analisa Diagnostica^® for colorimetric determination. Albumin:globulin ratio (A:G) was determined using the ALB and TPP values [Globulin = TPP − ALB; A:G = ALB:Globulin]. Glucose (GLU), triacylglycerol (TGL), and cholesterol (CHOL) concentration were determined by enzymatic method using commercial kits (Labtest Diagnóstica, Lagoa Santa, MG, Brazil).

Hematological and biochemical parameters were tested using one-way ANOVA and Tukey test for comparison of effects of different DP levels before and after challenges. To compare the different groups (non-stressed and stressed) data were subjected to a Kruskal-Wallis non-parametric test and complemented with a Mann-Whitney multiple comparisons test. Differences were considered to be significant at the 0.05 probability level. All analyses were performed after checking the normality of data. A cluster analysis was carried out on hemato-biochemical data of all samples including non-stressed and stressed fish to discriminate the different types of stress by proportion of similarity. The analyses were conducted using the software Minitab^® 16.1.1.0 (Minitab Inc., State College, Pennsylvania, USA).

Stress responses vary among fish species, leading to a wide range of physiological and biochemical mechanisms to overcome the situation or compensate the imbalance (Tort, 2011). Stress responses affect overall protein and energy metabolism, inducing mobilization of body energy reserves (Diógenes et al., 2019). A nutritionally complete diet can help fish to cope with stress, providing the nutrients and energy to support the stress response. Thus, fish nutritional history may have a protective effect counteracting some of the consequences of the stress response (Tort et al., 2004; Conceição et al., 2012).

In our study, we evaluated graded dietary DP levels under different types of stress and the capacity to maintain health standards. The stressors were chosen based on common aquaculture practices aiming to simulate both physical and environmental-related types of stress. As for environmental stressors, temperature-induced stress determined different hematological and metabolic responses. The main effect of CIS was on the leukogram and the main effect of HDOIS was on energy source and oxygenation.

Lymphopenia and neutrophilia were determined at CIS in all fish sampled, regardless the dietary DP levels. These results have been consistently observed in Nile tilapia, subjected to different stressors, irrespectively of the immunonutrition (Bly and Clem, 1992; Falcon et al., 2007; Barros et al., 2009; Fernandes Junior et al., 2010; Barros et al., 2014; Araújo et al., 2017). Although Nile tilapia has been described as a fish species that stands broad variation of temperature, its thermal comfort ranges from 27 to 30 °C (Boyd, 1990; El-Sayed, 2006). In the present study, a short period (30 h) under low temperature (17 °C) was enough to trigger the cell-mediated immune response. The neutrophilia observed was probably necessary to keep the fish healthy, since neutrophils are a critical component of the host defense system, forming the first line of cellular defense against invading organisms (Smith, 2000). Since MON count did not increase and no signs of bacteria were observed, we assumed that neutrophils were able to maintain health. Similar results were described by Bly and Clem (1992), Barros et al. (2009), and Araújo et al. (2017). Studies have shown that leukocyte numbers can vary according to intensity and time of stress. According to Tort (2011), acute stress determines a significant reduction in lymphocytes and monocytes and an increase in neutrophils, whilst a chronic stress determine an initial increase, followed by a general decrease in blood leukocyte numbers.

Conversely, under HDOIS, an increase in metabolism with higher demand for energy and oxygen were expected. Regardless of dietary DP levels, fish showed higher Hb values, although their values were within the normal range for healthy Nile tilapia (Hrubec and Smith, 2010; Damasceno et al., 2016; Xavier et al., 2020). Consequently, MCHC was generally higher, thus demonstrating higher hemoglobin concentration in the erythrocytes. Alterations in Hb and MCHC were expected because of the lower oxygen level under high water temperature. The absence of significance in RBC and Htc could be attributed to the length of the induced-stress period. The increase in Htc and MCV values under HDOIS, although not significantly different, can be considered beyond the normal range (Hrubec and Smith, 2010; Teixeira et al., 2012). Together, these results may suggest that immature RBC could have been released to counteract hypoxia induced by this stressor, maintaining tissue oxygenation (Fernandez and Grindem, 2000).

Even though no signs of bacteria were observed in Nile tilapia under HDOIS, fish fed 34% DP showed significant neutrophilia and monocytosis, which could be a result of the enhanced non-specific immune function. Arginine and phenylalanine levels above the dietary requirement (Diógenes et al., 2016) could have helped nitric oxide (NO) production, since arginine is the precursor for the biosynthesis of NO and phenylalanine can regulate NO production, being essential as a cofactor for NO synthase (Wu, 2013). Therefore, further studies assessing the innate immune response such as leukocyte activity and the production of oxygen and nitrogen reactive species could provide a better understanding on how the different stressors could affect fish physiology under challenging situations.

These results highlighted the importance of evaluating the thermal stress effect, even at higher water temperatures and longer periods, as it is commonly observed in tropical countries. Although no signs of bacteriosis were observed in the present study, the relationship between changes in water temperature and the incidence of bacteriosis has been previously reported (Kersters et al., 1996; Rodkhum et al., 2011).

Stress responses are energy-draining processes that uses triglycerides as the main energy source (Benton et al., 1994). Triglycerides are broken down into glycerol and fatty acids by lipase, and stress hormones contribute for lipolysis (Voet et al., 2002). Accordingly, the significant reduction observed in TGL under HDOIS (decrease in average 4.8-fold) supports the idea that Nile tilapia under high temperature requires extra energy to restore homeostasis. Under CIS, a significant decrease in TGL was also observed, but only in fish fed 32% DP (declining 2.17-fold). It is important to emphasize that, because Nile tilapia is a tropical fish species well-adapted to warm temperature (Zerai et al., 2010), under low temperature the metabolic rate decreases, and all physiological responses can be compromised. In CIS, fish fed the lowest and the highest DP levels showed significantly higher GLU, indicating stress, and the same occurred for fish fed the lowest DP level under HDOIS.

In summary, digestible protein ranging from 26 to 32%, at low temperature, and 26 to 34% at high temperature, seems to be able to keep fish homeostatic for a short term, probably as an energy-yielding nutrient. Indeed, under HDOIS, fish mobilize more TGL as energy source than in CIS. The diet formulated to contain 22% DP was not adequate to keep homeostasis, either at low or high temperature. Additionally, this diet did not meet arginine requirement, an important functional amino acid to synthesize nitric oxide, as aforementioned. According to Iwama et al. (2011), an imbalance in dietary protein concentration could impair the immune response and increase their susceptibility to diseases. Therefore, we believe that this level of DP was inadequate for meeting all physiological demands.

Transport and size-sorting are considered physical stressors that involves capturing, loading, transporting or size-sorting, unloading, and stocking. Fish subjected to these multiple-phase operations generally take a long time to recover and may suffer severe physiological consequences (Barton, 2002; Conte, 2004; Sutthi and Doan, 2020; Obirikorang et al., 2020). The production of glucose with stress is a source of energy for animals and a tool to help them cope with the increased energy demand (Balasch and Tort, 2019; Rudneva, 2013). Such condition, as highlighted in our study, can be confirmed by the increase in GLU and decrease in TGL after both physical stressors. Similar results were obtained for Nile tilapia under commercial condition after fish being subjected to size-sorting stress (Fernandes Junior et al., 2016).

As for oxygen demand, both stressors required high oxygenation, as evidenced by the overall increased MCHC and Hb values. Fish fed diets that meet their DP requirement for growth were able to maintain the reference values for healthy Nile tilapia erythrogram. In addition, alterations occurred in non-specific immune response leading to an increase in cell-mediated defense, mostly NEUT under TIS and SISS, and MON under SISS, mainly for DP levels below the nutritional growth requirement. The importance of protein on immune response has been previously described, since cytokines, immunoglobulins, and other components of the immune system are proteinaceous (Gatlin, 2002). Although the erythropoiesis was maintained under both SSIS and TIS, the oxygenation demand increased especially under deficient DP levels and TIS. This relationship between dietary DP levels and both red blood cells and hemoglobin syntheses suggest that fish increased Hb concentration rather than red blood cells number, since both are protein compounds and the dietary protein concentration was not even appropriate for growth. On the other hand, the white blood cells response was directly correlated with stress, independently of the dietary DP levels.

Cluster analysis was applied to each DP level to identify the induced-stress responses comparable to the non-stressing condition, considering all analyzed parameters. Among the stress conditions studied, SSIS and CIS were the most stressful for fish fed a dietary DP lower or equal to 32% DP. At 34% DP, CIS and TIS were the most stressful. On the other hand, HDOIS is clustered with non-stressing conditions. As it was previously mentioned, TIS and SSIS are considered multiple-phase-operations stress, associated with long time recovering and severe physiological consequences (Barton, 2002; Conte, 2004). Interestingly, HDOIS can be considered less stressful than CIS, probably because Nile tilapia is a tropical fish species well-adapted to warm temperature, as aforementioned. Due to the severity of SSIS and CIS, producers should avoid handling fish during low-temperature periods to reduce deleterious effects. Based on that, producers should consider meeting Nile tilapia protein requirement, especially the digestible concentration, aiming not just the growth performance, but also fish health, higher energy reserve, and better immune response.

Dietary protein does not mitigate stress response under SSIS and CIS; lymphopenia and neutrophilia are the main cell-mediated immune responses; dietary digestible protein below the nutritional requirement for Nile tilapia growth under SSIS and TIS impairs oxygenation; fish under HDOIS and SSIS demand more energy using triglycerides as an energy source; the diet formulated to contain 22% digestible protein is not adequate to keep homeostasis under temperature stress. Cluster analysis showed that, for digestible protein levels below the requirement for growth, SSIS and CIS are considered the most stressful conditions. At 34% digestible protein level, HDOIS response is comparable to that of non-stressing conditions.