Effects of Prolonged Exposure to Salinity on Blood and Immune Parameters of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Juveniles in Rearing Ponds

doi:10.21203/rs.3.rs-3797881/v1

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Effects of Prolonged Exposure to Salinity on Blood and Immune Parameters of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Juveniles in Rearing Ponds

https://doi.org/10.21203/rs.3.rs-3797881/v1

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Environmental and unfavourable circumstances significantly impact a fish's serum and blood biochemical parameters. The study examines the impact of long-term exposure to salinity stress on the blood and immune parameters of rainbow trout (Oncorhynchus mykiss) juveniles weighing 80 grams in farms with three different salinity levels in Golestan province (Iran): 0.5 ppt (freshwater), 3 ppt (brackish water), and 18 ppt (saltwater). After being purchased and transferred to the farms, the juveniles were fed a commercially available salmon diet for five months. After the trial period ended, blood samples were obtained from 60 healthy fish, with each fish weighing 250 grams at each farm. The samples were subjected to centrifugation in order to separate the serum from the blood cells. The desired parameters were measured using conventional laboratory techniques. The results revealed significant differences in the levels of white blood cells (WBCs) and immunoglobulin M (IgM) among the three experimental groups (p < 0.05). The fish in seawater showed the highest white blood cell count (14300 ± 310 mm³) and immunoglobulin M (0.428 ± 0.007 g/l) values. Red blood cell counts (RBCs), haematocrit (HCT), haemoglobin (Hgb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), complement C3 and complement C4 were all significantly lower in fish raised in brackish water compared to fish raised in freshwater and saltwater (p < 0.05). These parameters did not show any significant differences between fish reared in freshwater and saltwater (p > 0.05). The levels of total protein, glucose, cortisol, albumin, cholesterol, triglyceride, urea, uric acid, creatinine, alkaline phosphatase, lipase, and amylase were significantly higher in fish raised in saltwater compared to fish raised in fresh and brackish waters (p < 0.05). Significantly elevated levels of calcium, phosphorus, chloride, iron, copper, zinc, and magnesium ions were detected in saltwater fish that were raised (p < 0.05). Simultaneously, the amounts of sodium and potassium exhibited no statistically significant variations across all treatments (p > 0.05). To summarize, this research emphasizes that rainbow trout, a euryhaline species, exhibit better growth and survival rates when reared in water with a salinity of 18 parts per thousand (ppt). Furthermore, this species exhibits enhanced resistance to stressors at this specific salinity level, resulting in a more favourable habitat since these indicators tend to return to their original values observed in freshwater.

salinity

stress

haematology

blood serum

plasma ions

Oncorhynchus mykiss

Unlike controlled stress experiments conducted in a laboratory, rainbow trout juveniles in their natural habitat developed long-term salinity stress.
The study's findings highlight significant variations in the hematological and biochemical characteristics of rainbow trout raised under different salinity levels.
Biochemical analysis of rainbow trout serum unveiled notable distinctions in plasma ions among the various experimental conditions.
Furthermore, the analysis of rainbow trout serum indicated significant difference in immune parameters between the different experimental treatments.

Aquaculture plays a pivotal role in addressing global food shortages and malnutrition by providing a rich source of protein, vitamins, minerals, and amino acids. However, two key elements are indispensable to ensure sustainable aquaculture and optimal growth: healthy, disease-free fish and high-quality water. Achieving high production levels encompasses more than just the quantity and quality of feed; it also involves environmental factors (Al Hilali and Al-Khshali 2016; Fazio et al. 2013). Among these factors, salinity is one of the most influential, alongside temperature and dissolved oxygen, directly impacting fish growth (Mommsen 1998). Salinity can modify the energy available for fish growth through shifts in energy costs, such as ionic and osmotic adjustments. Nevertheless, predicting the intricate relationship between salinity and growth performance remains complex (Iwama 1996).

It is essential to recognise that the optimal salinity level varies for each fish species, affecting their growth and survival differently (Peyghan et al. 2013; Elarabany et al. 2017). The initial impact of salinity on fish is evident through osmotic adjustments. This process involves acquiring or losing plasma ions in response to varying salinity levels, allowing the maintenance of ion concentrations and internal body fluids. Organs responsible for osmotic regulation, such as the gills, kidneys, and intestines, play a crucial role in this process (Al Hilali and Al-Khshali 2016). Moreover, fluctuations in salinity can significantly affect a fish's physiological well-being (Kavya et al. 2016). These physiological changes trigger stress reactions, altering haematological compounds and immune mechanisms (Hamid et al. 2013).

Fish stress stems from physical and environmental changes, disrupting the delicate balance between the organism and its surroundings (Schulte et al., 2014). Stressors can perturb homeostasis (Elarabany et al. 2017), adversely impacting behaviour, growth, reproduction, immune functions, and disease tolerance (Kavya et al. 2016).

The global trend has long been shifting towards breeding fish adapted to salty and brackish waters, leading to significant expansion and diversity in this field (Ghanei Tehrani et al. 2017). Over half of aquaculture production is now dedicated to brackish and coastal waters (Partridge et al. 2008). Rainbow salmon (Oncorhynchus mykiss), a highly prized species with significant economic value in the aquaculture industry worldwide, thrives in various conditions and weight ranges, spanning fresh, brackish, and even salt water (Nafisi 2014).

Rainbow trout exhibit remarkable adaptability to rapid salinity changes, often transitioning seamlessly from freshwater to saltwater and thriving in such environments (Alizadeh et al. 2016). In recent years, numerous studies have focused on enhancing cultivation techniques and increasing yields for this species, examining water conditions at various salinities. Given the diverse and high-quality resources available, further research into internal saline water sources is imperative, considering each region's unique circumstances (Ghanei Tehrani et al. 2017).

Research by Tsintsadze (1991) revealed that rainbow trout exhibited the highest growth rates at salinities of 15–18% (the highest levels considered), while growth rates were lower at lower salinities, reaching the lowest in freshwater. Rassmussen and Ostenfeld (2000) observed optimal salmon growth at 15–18 g/l salinities. Altinok and Grizzle (2001) suggested that the suitable salinity level for this species is 9 g/l. Additionally, Finstad et al. (1988) reported that this fish, ranging from 40 to 120 grams, could tolerate salinity without displaying stress symptoms after a direct transfer from freshwater to saltwater. Jafaryan (2009) and Ghanei Tehrani et al. (2014) investigated O. mykiss ability to reproduce in waters with salinities of 2.95 and 14 ppt, respectively, and found better growth than freshwater fish.

Various studies have examined the physiological responses of fish to different salinity levels, revealing their impacts on parameters such as haematology and immune functions in species like tilapia (Oreochromus niloticus) (Elarabany et al. 2017; Küçük et al. 2013), common carp (Cyprinus carpio) (Al Hilali and Al-Khshali 2016), and grass carp (Ctenopharyngodon idella) (Makvandi et al. 2012). However, most of these studies have focused on laboratory conditions and short-term effects, with limited exploration of the long-term implications.

In conclusion, considering the constraints associated with freshwater resources and the burgeoning demand for aquaculture, it becomes imperative to shift the industry's foundation towards utilising non-freshwater sources, reducing reliance on rivers and freshwater. With its salinity approximately one-third that of the open seas (around 12–13 ppt), the Caspian Sea represents a dependable source for aquaculture development in coastal and even marine areas, particularly for certain species. Because rainbow trout are important to the aquaculture industry and there is not much information on how their immune systems change after being exposed to different salinities for a long time, in this study, we are going to look at the changes in specific biochemical parameters in serum and soluble ions in plasma of rainbow trout raised in brackish and saline waters and compare them to rainbow trout raised in freshwater.

Experimental design

The experiment spanned from November 2014 to April 2015, encompassing a five-month duration and employing conventional production techniques. This study unfolded across three distinct locations, each equipped with concrete ponds:

The Hammon cultivation fish farm, where salinity remained tightly regulated at 0.50 ± 0.02 ppt (freshwater, FW).
The Zaringol propagation fish farm maintained salinity at 3 ± 0.10 ppt (brackish water, BW).
The Gomishan shrimp farm boasts a salinity level of 18 ± 2.15 ppt.

The primary source of freshwater was a natural spring, while the brackish river facilitated operations at the brackish water farm. On the other hand, the Gomishan prawn breeding site nurtured O. mykiss juveniles in the waters of the Caspian Sea.

A total of 6,000 O. mykiss juveniles, each initially weighing 80 ± 5 g, underwent a 15-day acclimation period prior to the commencement of the study. Subsequently, these fish were relocated to aquaculture facilities featuring recirculating systems. During the rearing phase, the fish were fed a commercial meal, amounting to 3% of their body weight, thrice daily, between 9.00 a.m. and 7.00 p.m., until they reached satiety. The established light-dark cycle photoperiod adhered to a twelve-hour light and twelve-hour darkness schedule, coinciding with the feeding period.

To monitor water quality, we utilized a portable multi-parameter instrument (HQ40D18, Hach, Loveland, CO, USA). Detailed analytical information regarding water quality parameters is presented in Table 1.

Table 1

Water characteristics (mean ± SD) during the experiment and measurement of
Characteristic	Fresh Water	Brackish Water	Saline Water
Temperature (°C)	17.50 ± 1.24^a	17.08 ± 0.98^a	17.31 ± 1.56^a
Dissolved oxygen (mg/l)	7.24 ± 0.19^a	7.04 ± 0.54^b	6.79 ± 0.31^c
Conductivity (µs/cm)	1194.10±83.54^c	3150.22± 200.98^b	23920.57±2400.87^a
Salinity (ppt)	0.50±0.02^c	3 ± 0.1^b	18± 2.15^a
Total Hardness (mg/l)	328.45±49.35^c	448.35± 69.42^b	4160.43±498.28^a
Turbidity (NTU)	1.34 ± 0.15^c	2.21± 0.38b	3.15±0.67^a
TDS (mg/l)	583.25±134.37^c	897.46± 174.22^b	12640.59±2945.23^a
pH	7.60±0.32^c	7.82± 0.29^b	8.53± 0.36^a
Ca⁺⁺ Hardness (mg/l)	212.6±11.19^c	292.10± 16.98^b	2680.44±24.63^a
Mg⁺⁺ Hardness (mg/l)	116.7±5.24^c	156.14± 7.81^b	1480.22±73.49^a
Ca⁺⁺ (mg/l)	84.8±7.39^c	116.8±9.88^b	1072.63±16.47^a
Mg⁺⁺ (mg/l)	27.84±4.76^c	37.44±4.24^b	355.20±14.79^a
Na⁺ (mg/l)	116.6±10.21^c	196.4±19.63^b	3584.76±149.58^a
K⁺ (mg/l)	2.50±0.40^c	2.90±0.38^b	7.10±1.69^a

*Data are expressed as Mean ± S.D. Different letter in each column show significant difference between each hormone concentration (p < 0.05).

Blood sampling

Following the experimental phase, a 24-hour fasting period was established as a prerequisite for collecting fish samples. Each experimental cluster yielded a cohort of twenty fish, with an average weight of 250 ± 7.82g. These fish were then gently anesthetized by immersion in a solution containing 150 mg/l of Eugenia caryophyllata powder. The blood collection procedure involved the use of a syringe equipped with heparin and triazolyl. Blood was extracted from the caudal vein by inserting the needle perpendicular to the ventral surface of the fish, allowing blood to be drawn into the syringe. Each sample yielded 2–3 ml of blood volume. The collected blood samples were divided into two portions: approximately equal amounts of 1-1.5 ml were transferred to hemolysis tubes without anticoagulants for serum separation, and another 1-1.5 ml portion was placed in hemolysis tubes containing heparin (at a ratio of 10 microliters per 0.5 ml of blood). The plasma was then separated through centrifugation for 10 minutes at a force of 5000 ×g, utilizing the Denley BS400 centrifuge. Subsequently, the plasma samples were stored at a temperature of -20°C for hormonal assay estimation.

Haematology indices

Heparinized blood samples were employed for the assessment of hematological parameters. The quantification of white blood cell count, red blood cell count, and hemoglobin concentration followed the methods outlined by Blaxhall and Daisley (1973). Hematocrit percentage was determined using the England and Walford (1972) method. Additionally, corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated using the appropriate formulas as described by Blaxhall and Daisley (1973).

$$MCV\left(fl\right)=Ht \times 10/RBC$$

$$MCH\left(pg\right)=Hb\times 10/RBC$$

$$MCHC\left(\%\right)=Hb\times 10/Ht$$

Serum biochemical indices

Serum biochemical indices were assessed utilizing standard laboratory methods and commercial kits sourced from Pars Azmoun Company in Karaj, Iran, along with an autoanalysis device (Eurolyser, Iran), as outlined in Whicher (1996). Measurement of cortisol hormone levels was conducted using a cortisol hormone assay kit from Monobind in the USA, employing the direct ELISA method as described by Deane and Woo (2003).

Immunity indices

The complement pathway activity (C3 and C4) was assessed through the haemolysis of rabbit red blood cells, following the procedure outlined by Amar et al. (2000). Additionally, the measurement of immunoglobulin M (IgM) was conducted using the Bradford colorimetric method, as described by Kruger (1994).

Plasma ions

The monovalent sodium and potassium ion concentrations in heparin plasma were determined using a flame measurement method employing a film photometer (Jenway, PFP7 model, England). Additionally, the quantification of divalent ions such as calcium, magnesium, phosphorus, iron, copper, and zinc was performed through a colorimetric method utilizing commercial kits sourced from Pars Azmoun Company in Karaj, Iran, employing an autoanalyzer system. Chlorine measurement was conducted via a colorimetric method involving silver nitrate titration.

Statistical analysis

Statistical analyses were conducted using IBM SPSS software (Version 25, SPSS Inc., Chicago, IL, USA). The study employed a statistical approach, which entailed a one-way variance analysis (ANOVA) followed by a Duncan multiple range test (DMRT). Significance was set at a p-value below 0.05. This analysis aimed to discern variations among different experimental groups.

According to Table 1, trout cultured in brackish water exhibited a significantly higher white blood cell count compared to other experimental groups (p < 0.05). Conversely, the red blood cell count, haematocrit percentage, haemoglobin concentration, and mean haemoglobin in these fish were significantly lower (p < 0.05) compared to their counterparts in both fresh and saltwater. In the case of saltwater fish, their white blood cell count was significantly higher than that of freshwater fish (p < 0.05), but there was no significant difference in the red blood cell count, haematocrit percentage, haemoglobin concentration, or mean haemoglobin between freshwater and saltwater fish (p > 0.05).

Furthermore, the average globule volume in the blood of saltwater fish showed a significant increase (p < 0.05) compared to both freshwater and saltwater fish. However, there was no significant difference in this index between freshwater and brackish water fish blood (p > 0.05). Additionally, the average haemoglobin concentration measurements did not exhibit any significant differences among the experimental groups (p > 0.05).

Table 1

Comparsion of haematology parameters (mean ± S.D.) of rainbow trout in different salinity
Characteristic	Fresh Water	Brackish Water	Salt Water
WBC (Mil/UL)	12300.00± 250.00^c	14300.00± 310.00^a	13700.20± 210.00^b
RBC (Mil/UL)	0.96874± 0.03^a	0.8711± 0.02^b	0.9877± 0.02^a
Hemoglobin (g/l)	8.20± 0.20^a	6.80± 0.01^b	8.40± 0.02^a
Hematocrit (%)	24.20± 0.89^a	21.26± 0.69^b	24.90± 0.94^a
MCV (fl)	249.81± 8.49^b	241.65± 5.93^b	252.66± 9.44^a
MCH (pg)	84.65± 2.67^a	78.06± 3.22^b	85.05± 4.36^a
MCHC (%)	33.88± 1.12^a	32.38± 1.56^a	33.73± 1.87^a

Values are given as mean ± SD. Different letters denote significant differences (p < 0.05) between treatments.

The biochemical indices of rainbow trout blood serum were analysed, as presented in Table 2. The findings revealed that fish raised in saline water exhibited significantly higher levels of all the parameters in comparison to both freshwater and saltwater fish (p < 0.05).

Table 2

Comparison of biochemical parameters of blood serum of rainbow trout in different salinity
Characteristic	Fresh Water	Brackish Water	Salt Water
Blood Sugar (mg/dl)	42.00± 2.15^c	52.20± 4.12^b	77.20± 5.34^a
Cortisol (ng/ml)	227.12± 5.46^c	287.54± 7.66^b	589.23± 8.24^a
Creatinine (mg/dl)	0.38± 0.03^b	0.32± 0.02^c	0.85± 0.03^a
Uric Acid (mg/dl)	1.80± 0.09^b	2.50± 0.10^a	2.70± 0.08^a
Cholesterol (mg/dl)	144.35± 7.56^c	164.94± 7.44^b	311.41± 12.29^a
Triglycerides (mg/dl)	174.12± 9.22^b	145.37± 7.54^c	442.76± 12.62^a
Total Protein (g/dl)	3.42± 0.94^b	2.51± 0.57^c	6.38± 0.88^a
Albumin (g/dl)	0.98± 0.03^c	1.42± 0.08^b	3.1± 0.76^a
Alkaline Phosphatase (IU/L)	327.23± 14.66^b	139.65± 10.56^c	1210.41± 59.64^a
Amylase (U/L)	647.67± 37.81^c	738.54± 45.29^b	1595.47± 69.53^a
Lipase (U/L)	1.37± 0.03^c	0.23± 0.02^c	0.32± 0.01^c
Urea (mg/dl)	6.84± 0.12^c	8.27± 0.14^c	10.29± 0.26^c

Values are given as mean ± SD. Different letters denote significant differences (p < 0.05) between treatments.

Upon conducting a comparative analysis of the dissolved ion concentrations in the plasma, it was noted that there was no statistically significant difference in the levels of sodium and potassium ions among the experimental groups (p > 0.05). In contrast, the blood of saltwater fish displayed a significant increase in calcium, phosphorus, chloride, iron, copper, zinc, and magnesium ions in comparison to the other groups (p < 0.05). Notably, the highest concentrations of sodium and potassium were observed in fish reared in saltwater, as detailed in Table 3.

Table 3

Comparison of plasma ions rainbow trout reared at different salinities
Characteristic	Fresh Water	Brackish Water	Salt Water
Calcium (mg/dl)	9.90± 0.12^c	8.52± 0.19^b	16.30± 0.71^a
Phosphorus (mg/dl)	16.80± 0.88^b	13.12± 0.59^c	28.84± 0.87^a
Sodium (mEq/dl)	131.65± 4.99^a	130.36± 7.48^a	135.51± 8.23^a
Potassium (mEq/dl)	5.61± 0.34^a	5.63± 0.26^a	5.50± 0.62^a
Chloride (mEq/dl)	94.38± 5.81^b	94.325± 4.33^b	98.61± 5.46^a
Iron (micg/dl)	58.65± 1.07^b	60.28± 0.85^b	263.42± 13.53^a
Mg (mEq/dl)	3.19± 0.06^b	2.87± 0.08^c	6.54± 0.14^a
Cu (ng/ml)	101.33± 3.99^b	89.45± 5.67^c	121.63± 6.84^a
Zn (ng/ml)	494.62± 10.23^c	557.69± 12.88^b	710.37± 13.94^a

Values are given as mean ± SD. Different letters denote significant differences (p < 0.05) between treatments.

Comparing the immunity parameters revealed a significant decrease in C3 content (0.075 ± 0.01 mg/dl) (Fig. 1.a) and a concurrent increase in C4 content (0.297 ± 0.030 mg/dl) (Fig. 1.b) within the blood serum of fish cultivated in brackish water (p < 0.05). Conversely, there was no significant difference in these parameters between the blood of freshwater and saltwater-farmed fish (p > 0.05). Moreover, the IgM results demonstrated a significant disparity among all three experimental groups (p < 0.05). As salinity levels increased, the IgM content followed suit. The highest IgM levels (0.428 ± 0.007 g/l) were recorded in the blood of saltwater fish, whereas the lowest levels (0.248 ± 0.006 g/l) were found in freshwater fish blood (Fig. 1.c).

Salmons, specifically Oncorhynchus mykiss, are highly sensitive to environmental factors such as changes in pH, oxygen levels, temperature, crowding, and salinity. Among these factors, salinity changes can profoundly impact these fish's physiological and metabolic mechanisms. The osmotic stress experienced by fish in different water bodies can lead to significant changes in various biochemical parameters, and maintaining internal homeostasis requires considerable effort (Lambert et al. 1994; Gaumet et al. 1995; Zhang et al. 2017; Canosa and Bertucci 2023).

The ability to adapt salinity levels to enhance fish growth and well-being holds excellent potential for aquaculture practices. However, the biochemical variations observed and their potential effects on fish health and productivity necessitate further investigation. It is crucial to consider the broader ecological context and environmental variables to understand a fish's physiological and metabolic processes in response to salinity changes.

Our research showed that saltwater fish had significantly lower erythrocyte counts than other groups. This erythrocyte reduction was also seen in haemoglobin levels, haematocrit percentage, MCV, MCH, and MCHC. Girling et al. (2003) say that erythrocyte fragility decreases when cells lose water because of higher salinity.

Low salinity levels reduced haemoglobin and haematocrit concentration percentages (Soltanian and Fereidouni 2017). It is important to remember that Gabriel et al. (2007) hypothesized that abnormalities in erythropoiesis, the process of producing red blood cells, could also affect these outcomes.

The spleen plays a crucial role in erythropoiesis, and in response to heightened stress levels, specific organs, including the spleen, may swell, leading to increased production of red blood cells and haemoglobin. This response is believed to be triggered by elevated salt concentrations (Milligan and Wood 1982). Variations in the blood's aqueous phase composition could also contribute to erythrocyte count and haematocrit level elevation. Martinez-Alvarez et al. (2002) proposed that increased haematocrit levels could be due to reduced body water content resulting from fish being transferred to high-salinity aquatic environments and the differences in ion concentration between their internal and external surroundings.

However, it is essential to note that the initial increase in erythrocyte count and haemoglobin levels observed in response to hyperosmotic environments eventually returns to their initial values due to osmotic regulation mechanisms that maintain extracellular volume (Martinez-Alvarez et al. 2002). These findings support the observed tendency of haematological parameters in saltwater to revert to their measured levels in freshwater environments.

Jones and Randall (1978) noted that increased muscle activity could lead to elevated haemoglobin and haematocrit concentrations, along with the translocation of water from plasma to muscle. Yamamoto et al. (1980) and Wells and Weber (1990) also said that an increase in haemoglobin concentration and haematocrit percentage could make the spleen contract and release red blood cells that had been building up.

Research by Fazio et al. (2013) and Soltanian and Fereidouni (2017) on Mugil cephalus and Periophthalmus fish species, respectively, supported the idea that increased salinity levels result in reduced red blood cell count, haematocrit percentage, and haemoglobin concentration. Elarabany et al. (2017) researched tilapia fish (O. niloticus) and reported a significant decrease in these haematological parameters in response to different salinities (0, 4, 8, and 12 g/l) over 14 days.

Our study found no substantial differences between freshwater and saltwater habitats in the assessed parameters. This could be attributed to the adaptability of salmon to elevated salinities of up to 18 ppt and the tendency of these parameters to return to their normal levels in freshwater following acclimatization.

White blood cells play a vital role in assessing physiological stress in fish (Svobodova et al. 2001). Our investigation showed a significant reduction in white blood cell concentration in fish reared in brackish water compared to other experimental groups. The reduction in white blood cells, known as leukocytopenia, is a non-specific response of fish to environmental stress (Binuramesh and Michael 2011).

This reduction in white blood cells in response to low salinity levels can be attributed to the reduced tolerance of O. mykiss to such conditions. As a result, there is a decrease in leukocytes, which in turn leads to a decline in the organism's immune response. These findings align with previous research by Pourmozaffar et al. (2015), who reported a positive correlation between white blood cell count and increasing salinity levels up to 15 g/l.

However, it is worth noting that in the study of Sahafi et al. (2013), salmon raised in brackish water exhibited a significant increase in white blood cells compared to those raised in freshwater. Similarly, Amin (2014) observed elevated levels of white blood cells in Barbonymus gonionotus in brackish water.

Research on fish immune parameters related to salinity is an area that requires further investigation (Cuesta et al. 2005). Factors such as temperature, light, water quality, and salinity can significantly influence fish immunity and response (Magnadottir 2010). Some studies have suggested that salinity levels may increase immune parameters, specifically immunoglobulin M (IgM), in fish blood (Uribe et al. 2011). However, the underlying causes of these alterations are still not fully understood, and some propose that increased salinity levels may create a more conducive osmotic environment for pathogens (Bowden 2008).

Our study found that saltwater fish exhibited higher IgM levels, while freshwater fish had lower levels. This suggests a correlation between elevated salinity and increased blood IgM concentrations in rainbow trout. Yada et al. (2002) and Elarabany et al. (2017) reported similar findings in O. mossambicus and O. niloticus, respectively.

Additionally, we observed that elevated IgM levels in saltwater fish were associated with reduced complement C3 activity and increased C4 levels. The study did not find any statistically significant differences in complement activity between fish that live in freshwater and fish that live in saltwater. This suggests that the immune system's response to changes in salinity may involve different pathways than just a general rise or fall in complement activity.

These findings suggest that the salinity of the environment affects the immune response of rainbow trout. The higher IgM levels observed in saltwater fish suggest an enhanced adaptive immune response, possibly to counteract the higher pathogenic pressure associated with increased salinity.

The complement system plays a crucial role in innate immunity, and the observed changes in complement C3 and C4 levels could indicate alterations in the fish's ability to combat infections. Further research is needed to understand the specific mechanisms through which salinity influences the immune response of rainbow trout.

In conclusion, our study demonstrates that the salinity of their aquatic environment influences the haematological and immunological parameters of rainbow trout (Oncorhynchus mykiss). While some parameters, such as erythrocyte count and haemoglobin concentration, showed no significant differences between freshwater and saltwater fish, others, like white blood cell count and IgM levels, exhibited variations in response to salinity changes. The ability of rainbow trout to adapt to different salinity levels suggests a degree of physiological plasticity, allowing them to thrive in a range of aquatic environments. However, the specific mechanisms underlying these adaptations and their implications for fish health and immunity warrant further investigation. It is important to note that the results of this study provide valuable insights into the relationship between salinity and fish physiology. However, additional research is needed to fully understand the interplay between environmental factors, immune responses, and the overall health of rainbow trout and other fish species in various aquatic conditions.

WBCs

white blood cells

RBCs

red blood cells

Hgb

Haemoglobin

HCT

Haematocrits

MCV

mean corpuscular volume

MCH

mean corpuscular haemoglobin

MCHC

mean corpuscular haemoglobin concentration

IgM

Immunoglobulin M

Ethical Approval

not applicable

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Author Contribution

Mohammadreza Bivareh and Hojatollah Jafaryan wrote the main manuscript text and prepared figures. All authors reviewed the manuscript.

Availability of data and materials

The data that support the findings of this study are available from the corresponding

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Effects of Prolonged Exposure to Salinity on Blood and Immune Parameters of Rainbow Trout (Oncorhynchus mykiss (Walbaum, 1792)) Juveniles in Rearing Ponds

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Blood sampling

Haematology indices

Serum biochemical indices

Immunity indices

Plasma ions

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