Evaluation of gradual acclimatization of Litopenaeus vannamei post-larvae to freshwater in tropical regions: Emphasis on biological performance and physiological health responses

Evaluation of gradual acclimatization of Litopenaeus vannamei post-larvae to freshwater in tropical regions: Emphasis on biological performance and physiological health responses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of gradual acclimatization of Litopenaeus vannamei post-larvae to freshwater in tropical regions: Emphasis on biological performance and physiological health responses Herno Minjoyo, Suryadi Saputra, Maya Meiyana, Dwi Handoko Putro, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7191405/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vannamei shrimp ( Litopenaeus vannamei ) have long been cultivated in brackish or marine waters in Indonesia. However, production has faced challenges due to outbreaks of Vibrio parahaemolyticus . Research suggests that this pathogen does not thrive at very low salinity levels, prompting interest in low-salinity culture systems. To explore this approach, a study was conducted from March to April 2024 to evaluate the effects of gradual salinity reduction on the survival and physiological health of L. vannamei post-larvae (PL-10). The research was carried out at PT. Mochtar Sani Corporation (MSC), Campang Raya, Bandar Lampung, Indonesia. A Completely Randomized Design (CRD) was employed, consisting of four treatment groups, each replicated three times, namely Control K (Sudden salinity reduction from 10 ppt to 0.5 ppt); Treatment X ( Gradual salinity reduction of 2 ppt every 24 hours); Treatment Y (Gradual salinity reduction of 2 ppt every 48 hours); and Treatment Z (Gradual salinity reduction of 2 ppt every 60 hours).The highest survival rate was recorded in Treatment Y (89.0 ± 4.0%). Statistically, the control group exhibited a significantly lower survival rate (24.0 ± 6.0%) compared to all gradual reduction treatments (P 0.05). Additionally, the specific daily growth rate showed no significant differences among treatments, including the control group (P > 0.05). Physiological health indicators—such as hepatopancreas condition, lipid levels, and intestinal muscle ratio—remained within normal ranges throughout the acclimatization period, indicating that gradual salinity reduction did not negatively impact shrimp health. Aquaculture and Mariculture Litopenaeus vannamei Survival physiological health freshwater Introduction Litopenaeus vannamei , commonly known as vannamei or whiteleg shrimp, has become one of Indonesia’s most prominent aquaculture commodities. Since its introduction in 2001, this species has played a pivotal role in revitalizing the national shrimp industry and contributing to the diversification of fishery production (Hendarajat et al., 2007 ; Nababan et al., 2015 ; Sahabudin et al., 2020 ). Rising demand both domestically and internationally has driven farmers to adopt more intensive farming methods, typically involving high stocking densities (Supono & Wardiyanto, 2008; Bagus, 2021; Cahyanurani & Hariri, 2021 ; Cahyanurani & Dowansiba, 2022 ; Cahyanurani & Edy, 2022 ; Hidayat et al., 2019 ). Globally, P. vannamei is extensively cultivated across Central and South America, the United States, East and Southeast Asia, the Middle East, and parts of Africa (Benzie, 2009 ). In the United States, shrimp farming traditionally associated with marine or brackish environments has expanded inland, often just a few kilometers from the coast, utilizing both saltwater and freshwater sources. This shift marks a rapidly emerging sector within the aquaculture industry (Ednoff, 2001 ). The growth of inland shrimp farming in low-salinity—sometimes nearly freshwater—conditions is driven by factors such as the rising cost of coastal property, environmental limitations, and increasing domestic demand for shrimp (Hopkins et al., 1996 ). Pacific white shrimp ( Litopenaeus vannamei ) have demonstrated the ability to thrive in freshwater systems, with successful cultivation reported at total dissolved solids (TDS) levels as low as 650 ppm at the Harbor Branch Oceanographic Institution (Van Wyk et al., 1999 ; Van Wyk, 2013 ). Similar success has been achieved in low-salinity environments throughout other parts of the U.S. (Samocha et al., 1998 ; Ednoff, 2001 ; Samocha et al., 2002 ). Intensive shrimp farming systems, often characterized by limited environmental capacity, high stocking densities, overfeeding, and poor water quality management, can disrupt the ecological balance and increase shrimp susceptibility to disease (Zafar et al., 2015 ; Widodo et al., 2021 ). One innovative approach to mitigate disease outbreaks is the implementation of low-salinity culture systems (Ariadi et al., 2019 ; Ariadi et al., 2021 ; Panakorn, 2012 ). Among the major pathogens in Litopenaeus vannamei aquaculture are Vibrio species , particularly Vibrio parahaemolyticus , the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND). Monitoring Vibrio populations is considered a key indicator of shrimp health and culture conditions (Bauer et al., 2018 ; Pena et al., 2001). Schofield et al. ( 2021 ) showed that V. parahaemolyticus AHPND maintained its pathogenicity across a wide range of salinities but caused less mortality at lower salinities. As a halophilic bacterium, V. parahaemolyticus reproduces more effectively in high-salinity environments, producing higher levels of the pyrAB toxin under these conditions. Estrada-Pérez et al. (2020) investigated the performance of Litopenaeus vannamei during AHPND outbreaks across seven shrimp pond production cycles in Mexico between 2013 and 2016. As a euryhaline species, L. vannamei can adapt to a broad salinity range—from 2 to 40 ppt (Scabra et al., 2021 )—making it highly suitable for inland and low-salinity aquaculture systems (Maghfiroh et al., 2019; Tahe & Nawang, 2012 ). Low-salinity shrimp farming has been widely implemented globally (Roy et al., 2010 ), including in Indonesia, where it is practiced intensively in regions such as Lampung, Purworejo, Tuban, Lamongan, and Probolinggo (Ariadi et al., 2019 ). These systems typically maintain salinity levels below 10 g/L (Boyd & Thunjai, 2003 ). The benefits of low-salinity aquaculture include reduced disease incidence, lower toxicity from harmful gases, and inhibition of pathogenic microorganisms (Samocha et al., 2004 ; Supono, 2019 ; Valencia-Castañeda et al., 2019 ). From both ecological and economic perspectives, the development of low-salinity vannamei shrimp farming holds strong potential for sustainable aquaculture expansion (Ariadi et al., 2019 ). Post-larval Litopenaeus vannamei often experience significant stress and mortality when transferred from acclimation tanks to freshwater or low-salinity solutions with markedly different ionic compositions. Such mortality, frequently occurring within 24 hours of transfer, is commonly attributed to excessively rapid acclimation—particularly when salinity decreases by more than 50% within an 8-hour window (Van Wyk et al., 1999 ). Additional contributing factors include ionic imbalances in the destination water (McGraw & Scarpa, 2003 ) and the lack of an appropriate transitional phase during the transfer process. Currently, there is limited information regarding the minimum ionic requirements necessary for marine shrimp, such as Litopenaeus vannamei , to survive in freshwater environments containing less than 1000 ppm total dissolved solids (TDS), as well as a lack of standardized protocols for safe freshwater acclimatization (Van Wyk et al., 1999 ; Anonymous, 2002 ; McGraw & Scarpa, 2002 ; McGraw et al., 2002 ; McGraw & Scarpa, 2003 ). A more comprehensive understanding of the factors that affect shrimp survival during salinity transitions—particularly the acclimation rate—is essential to support the sustainable development of freshwater-based L. vannamei aquaculture. This study aimed to evaluate the biological performance and physiological health of vannamei shrimp subjected to salinity reductions from seawater or high-salinity conditions to freshwater in tropical environments. Material and methods Test animals The test animals in this study were Litopenaeus vannamei post-larvae (PL-10), initially maintained at a salinity of 10 ppt and a temperature range of 30–31.5°C at the hatchery. A total of 1,200 shrimp were sourced from a commercial hatchery located at Kalianda Resort, South Lampung, Indonesia. The shrimp were transported in sealed containers to the research facility, with a transit time of approximately one hour. Research procedures, data collection and research design This research was carried out at a freshwater Litopenaeus vannamei shrimp cultivation facility located in Satmakura Campang, Bandar Lampung, Indonesia. The experimental setup consisted of 12 aquaria, each with a total capacity of 120 liters and filled with 100 liters of water and was stocked with 100 shrimps every container. Each aquarium was equipped with an aeration system and a recirculating water filtration unit to ensure optimal water quality throughout the study. The materials used in this study included Litopenaeus vannamei post-larvae (PL-10) maintained at a salinity of 10 ppt, commercial shrimp feed containing 45–46% protein, freshwater, seawater, and mineral supplements. At the start of the experiment, the salinity of the culture medium was adjusted to match the hatchery conditions (10 ppt). Throughout the study, the shrimp were fed artificial feed with a protein content of 45–46%, administered four times daily at ad libitum levels. Acclimatization to lower salinity levels (desalination) was carried out gradually until the target salinity of 0.5 ppt was achieved. This was done by periodically removing water from the containers and replacing it with freshwater, with the volume calculated to reach the intended reduction rate. This method aligns with the findings of McGraw & Scarpa ( 2004 ), who reported improved survival rates when the acclimatization period exceeded 72 hours. The experiment employed a Completely Randomized Design (CRD) consisting of four desalination treatments, each with three replicates: (1) Control (K) — a drastic reduction from 10 ppt to 0.5 ppt; (2) Treatment X — gradual reduction of 2 ppt every 24 hours; (3) Treatment Y — gradual reduction of 2 ppt every 48 hours; and (4) Treatment Z — gradual reduction of 2 ppt every 60 hours. Observations of growth and survival performance in post-larval whiteleg shrimp ( Litopenaeus vannamei ) were carried out during the desalination acclimatization process, which involved a gradual reduction in salinity from 10 ppt to 0.5 ppt. In addition to these performance metrics, physiological health parameters were assessed, including hepatopancreas condition, lipid content, and the gut muscle ratio (GMR). These evaluations were conducted on 10% of the shrimp sampled from each replicate. The percentage of hepatopancreas tubules, lipid content, and GMR were measured as indicators of internal physiological health. Microbiological analyses were also conducted, including total bacterial counts (TBC) in the culture water and total Vibrio counts (TVC) in both the water and shrimp samples. These measurements were taken from each replicate to assess the microbial load of the culture environment and the potential presence of pathogenic bacteria. Water quality parameters—including dissolved oxygen (DO), temperature, pH, salinity, conductivity, and total dissolved solids (TDS)—were measured daily in the morning using a multiparameter water quality meter (Merck AZ Instrument, Model 86031). Alkalinity was analyzed according to standard methods prescribed by the American Public Health Association (APHA, 2017). Ammonia and nitrite concentrations were quantified using a UV/VIS spectrophotometer (Perkin Elmer Lambda 35), following the Indonesian National Standards: SNI 06-6989.30-2005 for ammonia and SNI 06-6989.9-2004 for nitrite. To maintain optimal environmental conditions, mineral analysis was also performed. The concentrations of magnesium (Mg²⁺), potassium (K⁺), and calcium (Ca²⁺) were determined using an atomic absorption spectrophotometer (AAS), based on the ASTM E663-86 standard and the operational procedures of the Shimadzu AA-7000. Physiological response parameters Total hemocyte count (THC) analysis Stress levels in Litopenaeus vannamei were evaluated by analyzing hemocyte samples collected on day 0 (baseline) and at the end of the study. The Total Hemocyte Count (THC) was measured following the method described by Blaxhall and Daisley (1973). All analyses were conducted at the Laboratory of Lampung University. Data analysis Data on THC concentration, survival rate, specific daily growth rate, Total Vibrio Count (TVC), Total Bacterial Count (TBC), and the TVC/TBC ratio were analyzed using one-way ANOVA at a 95% confidence level. When significant differences were detected among treatments, a Least Significant Difference (LSD) post hoc test was conducted to identify specific group differences. All statistical analyses were performed using SPSS version 28. In contrast, water quality parameters were analyzed descriptively. Results Survival rate The survival rate of Litopenaeus vannamei exposed to a gradual reduction in salinity from 10 ppt to 0.5 ppt varied significantly across treatments. In the control group (K), which experienced an abrupt salinity drop from 10 ppt to 0.5 ppt, the survival rate was notably low at 24.0 ± 6.0%. In contrast, the gradual acclimatization treatments demonstrated markedly higher survival rates: 83.7 ± 8.5% in treatment X (2 ppt reduction every 24 hours), 89.0 ± 4.0% in treatment Y (2 ppt reduction every 48 hours), and 85.0 ± 22.0% in treatment Z (2 ppt reduction every 60 hours). One-way ANOVA revealed that the survival rates in treatments X, Y, and Z were significantly higher than that of the control group (P 0.05) in absolute weight gain or length increase among all treatments, including the control group. Similarly, the specific daily growth rate (SGR) showed no significant variation across treatments (Table 1 ). The lack of significant differences in growth parameters may be attributed to the relatively short duration of the experimental period. Table 1 Biological parameters of vannamei shrimp after salinity reduction process Parameter Treatment K (control) X Y Z Life sustainability (%) 24.0 ± 6.0 a 83.7 ± 8.5 b 89.0 ± 4.0 b 85.0 ± 22.0 b Daily growth rate (%/day) 1 0.4 ± 1.29 a 1 0.3 ± 2.25 a 1 1.6 ± 0.9 3 a 1 5.9 ± 4.69 a Note: Different superscripts in the same row indicate significant differences between treatments (p < 0.05); K = control ( drastic decrease in salinity from 10 ppt to 0.5 ppt); X = decrease of 2 ppt every 24 hours; Y = decrease of 2 ppt every 48 hours; Z = decrease of 2 ppt every 60 hours Physiological response Observations of the Total Hemocyte Count (THC) in Litopenaeus vannamei post-larvae during salinity reduction from 10 ppt to 0.5 ppt revealed distinct physiological responses across treatments. In the control group, THC declined from the initial baseline of 75 ± 0.0 × 10⁵ cells/mL to 63 ± 2 × 10⁵ cells/mL. Treatment X showed a further decrease to 58.5 ± 3.5 × 10⁵ cells/mL, while treatment Z recorded a slightly higher value of 62.5 ± 5.5 × 10⁵ cells/mL. Notably, treatment Y exhibited an increase in THC to 77.5 ± 8.5 × 10⁵ cells/mL, surpassing the baseline level. Statistical analysis using one-way ANOVA indicated a significant difference (P < 0.05) between treatment Y and the other groups (control, X, and Z), suggesting that the gradual salinity reduction protocol of 2 ppt every 48 hours (treatment Y) may offer the most favorable conditions for maintaining hemocyte stability (Table 2 ). Table 2 Physiological response of Litopenaeus vannamei l arvae after salinity reduction process Parameter Treatment K (control) X Y Z Initial THC body (x 10 5 cells /mL) 75 ± 0.0 a 75 ± 0.0 a 75 ± 0.0 a 75 ± 0.0 a Final THC body (x 10 5 cells /mL) 63 ± 2 a 58.5 ± 3.5 a 77.5 ± 8.5 b 62.5 ± 5.5 a Note: Different superscripts in the same row indicate significant differences between treatments (p < 0.05); THC = Total Hemocyte Count; K = control by drastically reducing salinity from 10 ppt directly to 0.5 ppt); X = decrease of 2 ppt every 24 hours; Y = decrease of 2 ppt every 48 hours; Z = with decrease of 2 ppt every 60 hours Shrimp health condition The outcomes of gradually reducing salinity from 10 ppt to 0.5 ppt in Litopenaeus vannamei are presented in Table 3 . There were no significant differences (P > 0.05) between the treatment groups and the control in terms of hepatopancreas structure, tubule integrity, lipid content within the hepatopancreas, or the gut muscle ratio (GMR) at the end of the acclimation period. These results suggest that gradual salinity reduction does not negatively impact the internal physiological health of the shrimp. Further evaluation revealed a marked decrease in the total Vibrio count (TVC) in the culture water across all treatments compared to baseline levels. However, statistical analysis showed no significant differences in TVC between the treatment groups and the control at the end of the study (P > 0.05). In contrast, the total bacterial count (TBC) in the water exhibited a significant reduction (P < 0.05) in the treatment groups, particularly in treatments Y and Z, which demonstrated a substantially lower microbial load than the control. Although both Vibrio and total bacterial counts declined over the course of the study, the final ratio of Vibrio to total bacteria did not show significant differences among the treatment and control groups (P > 0.05), as detailed in Table 3 . These results suggest that a gradual reduction in salinity may effectively suppress the overall bacterial population, potentially enhancing water quality and promoting better shrimp health. At the end of the study, assessments of Litopenaeus vannamei tissue revealed a significant decrease in both Vibrio and total bacterial counts across all treatments and the control. However, statistical analysis indicated no significant differences in total bacterial load between the treatments and the control group (P > 0.05). In contrast, the Vibrio count within shrimp tissue was significantly lower in treatment X compared to the control (P 0.05). Additionally, the ratio of Vibrio to total bacteria in shrimp tissue declined across all treatments and the control group (Table 3 ). The most substantial reduction was observed in treatment X, where the Vibrio -to-total bacteria ratio was significantly lower than that of the control (P 0.05). These results indicate that while gradual salinity reduction can effectively lower both overall bacterial levels and Vibrio counts in shrimp tissue, treatment X may be particularly effective in suppressing potentially pathogenic Vibrio populations during the acclimatization to freshwater. Table 3 Health parameters, TVC and TBC of vannamei shrimp after salinity reduction from 10 to 0.5 ppt Parameter Beginning Treatment K X Y Z hepatopancreatic tubules ( %) 72.55 ± 1.52 84.13 ± 8.25 a 74.15 ± 5.1 1 a 85.12 ± 3.25 a 83.15 ± 5.15 a hepatopancreatic lipid (%) 73.45 ± 0.55 81.08 ± 6.33 a 83.55 ± 5.55 a 85.53 ± 6.15 a 82.35 ± 10.25 a GMR (Intestinal muscle ratio) 2.55 ± 0.05 2.65 ± 0.25 a 2.58 ± 0.05 a 2.59 ± 0.15 a 2.52 ± 0.05 a TVC Water ( CFU mL − 1 ) 26.0 ± 0.0 6.5 ± 1.5 a 2.5 ± 1.5 a 12.0 ± 6.81 a 7.5 ± 2.5 a Air TB (CFU mL − 1 ) 368.0 ± 0.0 324.5 ± 29.5 a 410.5 ± 50.5 a 550.0 ± 60.0 bc 789.0 ± 109.0 d TVC/TBC Ratio of Water (%) 9.73 ± 0.0 2.04 ± 0.65 a 0.58 ± 0.30 a 2.18 ± 0.98 a 0.99 ± 0.46 a Body TB (CFU mL − 1 ) 2240.0 ± 0.0 967.5 ± 90.5ᵃ 816.0 ± 8.0ᵃ 906.5 ± 261.5ᵃ 855.5 ± 121.5ᵃ TVC Body (CFU mL − 1 ) 288.0 ± 0.0 9.33 ± 15.31ᵃ 53.33 ± 14.50ᵇ 5.0 ± 1.0ᵃ 11.5 ± 6.50ᵃ TVC/TBC Body Ratio (%) 12.86 ± 0.00 0.89 ± 1.44ᵃ 6.90 ± 2.29ᵇ 0.56 ± 0.05ᵃ 1.44 ± 0.97ᵃ Note: Different superscripts in the same row indicate significant differences between treatments (p < 0.05); TVC = Total Vibrio Count; TBC = Total Bacterial Count; K = control ( drastically decreasing salinity from 10 ppt to 0.5 ppt); X = decreasing salinity by 2 ppt every 24 hours; Y = decreasing salinity by 2 ppt every 48 hours; Z = decreasing salinity by 2 ppt every 60 hours Water quality parameters Throughout the study, salinity was gradually reduced from 10 ppt to 0.5 ppt during the rearing of Litopenaeus vannamei , with careful monitoring of water quality parameters. Overall, the recorded parameters—particularly temperature, dissolved oxygen (DO), pH, and alkalinity—remained within acceptable ranges to support shrimp survival and health (Table 4 ). However, nitrite levels were consistently elevated across all treatments, potentially posing a health risk. In addition, concentrations of key minerals such as calcium, magnesium, and potassium were relatively low, which could negatively impact osmoregulatory function and general physiological performance. Table 4 Water quality and mineral parameters after salinity reduction from 10 to 0.5 ppt Parameter Beginning Treatment K X Y Z pH 8.19 ± 0.19 7.94 ± 0.36 a 8.19 ± 0.19 a 8.05 ± 0.35 a 7.92 ± 0.41 a Temperature ( 0 C) 30.89 ± 0.20 30.89 ± 0.20 a 30.45 ± 0.07 a 30.33 ± 0.05 a 31.16 ± 0.11 a DO ( mg L − 1 ) 4.56 ± 0.04 4.56 ± 0.04 a 4.81 ± 0.2 a 4.76 ± 0.6 a 4.58 ± 0.04 a Alkalinity ( mg L − 1 ) 143.0 ± 0.0 58.5 ± 2.5 a 56.0 ± 0.0 a 54.0 ± 2.0 a 51.5 ± 4.5 a Nitrite ( mg L − 1 ) 0.0 ± 0.0 1.85 ± 0.05 a 1.84 ± 0.04 a 1.86 ± 0.04 a 1.87 ± 0.003 a Ammonium ( mg L − 1 ) 0.05 ± 0.02 0.24 ± 0.02 a 0.05 ± 0.02 a 0.19 ± 0.14 a 0.09 ± 0.001 a Conductivity (µS cm − 1 ) 39.25 ± 0.25 1,129 ± 160.5 a 1081.67 ± 255 a 975 ± 76 a 1,059.3 ± 173 a TDS (mg L − 1 ) 19.4 ± 0.4 564.0 ± 81 a 613.67 ± 180 a 487.33 ± 37 a 529.25 ± 55 a Calcium (mg L − 1 ) 76.58 ± 0.00 0.05 ± 0.02 a 0.05 ± 0.002 a 0.19 ± 0.27 a 0.03 ± 0.01 a Magnesium (mg L − 1 ) 102.9 ± 15.58 0.14 ± 0.07 a 0.20 ± 0.09 a 0.14 ± 0.08 a 0.15 ± 0.06 a Potassium (mg L − 1 ) 28.34 ± 1.07 0.17 ± 0.02 a 2.59 ± 0.25 a 1.43 ± 0.86 a 1.00 ± 1.51 a Note: Different superscripts in the same row indicate significant differences between treatments (p < 0.05); K = control (drastically decreasing salinity from 10 ppt to 0.5 ppt); X = decreasing salinity by 2 ppt every 24 hours; Y = decreasing salinity by 2 ppt every 48 hours; Z = decreasing salinity by 2 ppt every 60 hours Discussion This study assessed the effects of gradual salinity reduction on the survival of Litopenaeus vannamei post-larvae (PL-10), using a reduction rate of 2 ppt implemented at three different time intervals, along with a control group that experienced an abrupt drop in salinity from 10 ppt to 0.5 ppt. The results revealed significant differences in survival rates between the control and all gradual acclimation treatments (X: 24-hour interval; Y: 48-hour interval; Z: 60-hour interval). The survival rates observed in this study were notably higher than those reported by McGraw et al. ( 2002 ), who recorded only 5% and 13% survival at 48 and 24 hours, respectively, for PL-10 shrimp acclimated to 1 ppt. In comparison, this study achieved survival rates of 83.7 ± 8.5% (24 h), 89.0 ± 4.0% (48 h), and 85.0 ± 22.0% (60 h), aligning more closely with the survival performance typically observed in older post-larvae (PL-15–PL-20) in previous research. William et al. (2004) also reported comparable survival outcomes in PL15 shrimp acclimated to low salinity with added mineral ions. Notably, the survival rates observed in the present study were slightly higher than those documented by Saputra et al. ( 2024 ), who conducted acclimation at a final salinity of 0.5 ppt, and Abrori et al. ( 2022 ), who used 5 ppt. McGraw and Scarpa ( 2004 ) found that acclimation duration and habituation period did not significantly impact 24-hour shrimp survival, which ranged from 76–81% and 73–82%, respectively. However, other studies indicate that gradual acclimation enhances survival outcomes. Jayasankar et al. ( 2009 ) demonstrated near-total survival of P. vannamei acclimated to 5 ppt through a stepwise salinity reduction protocol, while McGraw and Scarpa ( 2004 ) further showed improved survival at 1 ppt when the acclimation period was extended to 72 hours. The osmoregulatory capacity of crustaceans serves as a reliable indicator of their physiological condition and acts as a sensitive biomarker for evaluating the effects of environmental stressors, pollutants, or pathogens (Lignot et al., 2000 ). These organisms are capable of maintaining internal osmotic equilibrium, and fluctuations in this ability may signal physiological stress. Regarding growth performance, no significant differences were observed between the treatment groups and the control. Nonetheless, treatment Z exhibited a marginally higher daily growth rate compared to the other treatments. Overall, the growth outcomes in this study were superior to those reported by Saputra et al. ( 2024 ), suggesting that the salinity reduction protocol applied—particularly in treatment Y—closely approached an optimal acclimatization strategy. This likely reflects a physiological equilibrium between the shrimp’s internal regulatory systems and their external environment. These findings align with Saputra et al. ( 2024 ), who similarly reported that gradual salinity reduction did not significantly impact growth. From a physiological standpoint, the total hemocyte count (THC) in L. vannamei PL-10 declined following salinity reduction from 10 ppt to 0.5 ppt relative to initial values. Nonetheless, statistical analysis revealed no significant differences in THC across treatments and the control, with the exception of treatment Y (77.5 ± 8.5 × 10⁵ cells/mL), which also yielded the highest survival rate (89.0 ± 4.0%). These findings suggest that the carefully managed salinity reduction protocol implemented in treatment Y contributes to maintaining physiological homeostasis and enhancing survival in whiteleg shrimp post-larvae. Physiological responses of Litopenaeus vannamei to reduced salinity during acclimatization were assessed by examining hepatopancreatic tubule structure, lipid content within the hepatopancreas, and the gut muscle ratio (GMR) before and after salinity reduction. The analysis revealed no statistically significant differences between the treatment groups and the control. These findings are consistent with those reported by Saputra et al. ( 2024 ). The hepatopancreas is a critical organ in shrimp, essential for digestion and metabolic regulation (Wang et al., 2023). The consistent stability of this organ across all treatments suggests that the gradual acclimation protocol successfully minimized osmoregulatory stress, thereby preserving its functional integrity. This maintenance of hepatopancreatic function is crucial for supporting efficient metabolic and digestive processes during periods of salinity reduction. Huang et al. ( 2019 ) emphasized the role of lipids—particularly triglycerides and phospholipids—in shrimp osmoregulation, noting their function as key energy reserves and their importance in sustaining physiological activity and cell membrane stability under changing environmental conditions. In this study, the gut muscle ratio (GMR)—a key indicator of digestive efficiency and muscle development—remained consistent across all treatment groups (Table 4 ). As highlighted by Peregrino (2006), the relative proportion of muscle to gut serves as a reliable metric for evaluating postlarval quality. These results suggest that gradual salinity reduction did not negatively impact the digestive system or overall growth of L. vannamei . The acclimatization from 10 ppt to 0.5 ppt was well tolerated and appears to support physiological resilience. This aligns with findings from Lin and Chen ( 2001 ), who demonstrated the species’ robust osmoregulatory capacity across a broad salinity range, enabling metabolic equilibrium and sustained health under salinity stress. Similar observations were also reported by Saputra et al. ( 2024 ), further reinforcing the effectiveness of gradual salinity adjustment. One of the primary contributors to disease outbreaks in Litopenaeus vannamei aquaculture in Indonesia is Vibrio parahaemolyticus , a pathogenic bacterium known for its rapid proliferation in high-salinity conditions, including brackish water systems. In this study, observations—particularly at the conclusion of the trial period—revealed a substantial reduction in total bacterial count (TBC) within both the rearing water and the shrimp, relative to initial measurements. This decline was accompanied by a marked decrease in total Vibrio count (TVC) in both the culture water and shrimp samples. Similar trends were reported by Saputra et al. ( 2024 ), who also documented a reduction in total Vibrio count (TVC) in shrimp tissue across various salinity treatments, although inter-treatment variability was minimal. Conversely, total bacterial count (TBC) in the culture water increased in all treatment groups—except the control—by the end of the study period. This rise is likely attributable to the accumulation of organic matter, including uneaten feed, metabolic byproducts, and shrimp fecal matter, as previously highlighted by Seethalakshmi et al. ( 2021 ). Despite the increase in total bacterial count (TBC) in the culture water, the ratio of total Vibrio count (TVC) to TBC in both the culture medium and shrimp tissue declined significantly, serving as a positive indicator of shrimp health. Gradual and carefully controlled salinity reduction appears to play a vital role in maintaining physiological homeostasis in L. vannamei (Lin & Chen, 2001 ), thereby reducing vulnerability to microbial infections. Effective acclimatization also supports immune system stability during environmental transitions (Chang et al., 2024 ). As salinity approached freshwater levels, the prevalence of Vibrio parahaemolyticus —which thrives under higher salinity conditions—was notably suppressed. These findings are consistent with Saputra et al. ( 2024 ), who reported that gradual, non-abrupt salinity adjustments reduce physiological stress and mitigate the risk of pathogenic bacterial infections, particularly those caused by Vibrio spp. Analysis of total Vibrio count (TVC) and total bacterial count (TBC) in the bodies of Litopenaeus vannamei at the conclusion of the acclimation period revealed a significant reduction in both bacterial populations. This decline underscores the positive impact of a carefully managed, gradual salinity reduction in promoting shrimp health by limiting the prevalence of pathogenic bacteria such as Vibrio parahaemolyticus , thereby lowering the risk of infection and disease. While TBC decreased significantly across all groups—including the control—no statistically significant differences were detected among treatments. In contrast, TVC exhibited a significant decline, particularly in treatment X, which also displayed a notable change in the TVC/TBC ratio within shrimp tissue. Although these results differ slightly from those documented by Saputra et al. ( 2024 ), they are in alignment with the broader conclusions presented by Chang et al. ( 2024 ), which emphasize the effectiveness of controlled acclimation protocols in mitigating microbial threats. Simultaneously, concentrations of key minerals—magnesium (Mg²⁺), calcium (Ca²⁺), and potassium (K⁺)—declined significantly following salinity reduction to 0.5 ppt (Table 4 ). This decrease is likely linked to the increased utilization of these ions by shrimp to support critical physiological functions, such as osmoregulation and exoskeleton development. Magnesium plays a central role in maintaining ionic equilibrium and is essential for exoskeletal structure (Davis et al., 2002 ). As demonstrated by Cheng et al. ( 2006 ), shrimp actively extract minerals from their environment to counter osmotic challenges in low-salinity conditions. Potassium, vital for sustaining intracellular ion gradients, osmotic pressure, and neuromuscular activity, is similarly absorbed at elevated rates during hypoosmotic stress (Roy et al., 2010 ; Huong et al., 2010). Calcium is another essential element, particularly important for molting, and its decreased levels reflect increased physiological demand during acclimation—a trend consistent with observations by Davis et al. ( 2002 ). Fluctuations in mineral concentrations within aquaculture systems play a vital role in sustaining the ecological balance of the culture environment. These minerals are not only critical for the physiological well-being of shrimp but also serve as essential nutrients for the growth and metabolism of aquatic microorganisms (Zhang et al., 2020 ). The decline in mineral levels observed during salinity acclimatization is influenced both by increased mineral uptake by shrimp and the biological activity of resident microbial communities. To counter these reductions and maintain optimal shrimp performance, mineral supplementation—specifically with magnesium, calcium, and potassium—is essential throughout the salinity reduction process. Previous studies have demonstrated that such supplementation enhances shrimp survival and growth during transitions to low-salinity conditions (Saputra et al., 2024 ). Wang et al. (2023) further emphasized that targeted mineral enrichment improves osmoregulatory capacity, thereby promoting metabolic stability and facilitating successful adaptation under hypoosmotic stress. During this study, the gradual reduction of salinity from 10 ppt to 0.5 ppt was accompanied by measurable shifts in water quality parameters (Table 4 ). Despite these changes, dissolved oxygen (DO) levels consistently remained within the optimal range, ensuring sufficient support for the physiological requirements and post-larval development of Litopenaeus vannamei . The observed DO values satisfied the oxygen demands necessary for aerobic respiration, a critical component of growth and molting processes. Water temperature across all treatment groups also remained within the optimal spectrum, ranging from 30.89 ± 0.20°C to 31.16 ± 0.11°C. These findings align with the work of Venkateswarlu et al. ( 2019 ), who identified 30°C as the ideal thermal condition for L. vannamei . Together, stable temperatures and adequate DO levels fostered a conducive environment for metabolic function and physiological stability throughout the acclimation period. The application of a consistent and accurately managed salinity reduction protocol—targeting a final salinity of 0.5 ppt—proved effective in alleviating osmotic stress in Litopenaeus vannamei . Maintaining stable salinity conditions is critical for minimizing the risk of osmotic shock, which can significantly compromise shrimp health and survival during transitions from high-salinity to freshwater environments. Chen et al. (2015) demonstrated that a gradual acclimatization strategy enables post-larval shrimp to adjust more efficiently to salinity shifts, thereby mitigating the physiological stress linked to osmoregulatory demands. Following the salinity reduction, conductivity and total dissolved solids (TDS) levels declined significantly across all treatment groups (Table 4 ). These changes reflect a reduced concentration of dissolved ions and particulates in the culture water. Conductivity values were notably lower in all treatments compared to the control, indicating diminished ion availability. Adequate levels of dissolved ions are essential for efficient osmoregulation in L. vannamei , particularly under conditions of fluctuating salinity (Duan et al., 2022 ). The observed decline in conductivity and TDS suggests that the shrimp were able to physiologically adapt to low-salinity conditions without disruption to metabolic function, despite shifts in water chemistry. These results are consistent with findings by Saputra et al. ( 2024 ), who emphasized the value of gradual mineral and salinity adjustments in supporting shrimp adaptation during environmental transitions. The implementation of a controlled and consistent salinity reduction protocol—reaching a final concentration of 0.5 ppt—effectively mitigated osmotic stress in Litopenaeus vannamei . Maintaining stable salinity levels is essential in preventing osmotic shock, which can severely impact shrimp health and survival during transitions from saline to freshwater environments. Supporting these findings, Chen et al. (2015) demonstrated that gradual acclimatization enhances the ability of post-larval shrimp to cope with salinity fluctuations, thereby reducing the physiological burden associated with osmoregulatory adjustment. Alkalinity was a key water quality parameter evaluated in this study, and results indicated that levels remained relatively stable across all treatment groups. This consistency suggests that the shrimp experienced minimal acid-base disruption throughout the acclimatization process. Stable alkalinity is essential for preserving acid-base homeostasis, which underpins effective osmoregulatory function and nitrogen excretion—two critical physiological processes influencing the survival of Litopenaeus vannamei during cultivation (Zhang et al., 2016 ; Zhang et al., 2023 ; Saputra et al., 2024 ). Fluctuations in ammonia and nitrite concentrations throughout the study reflected ongoing biological processes within the culture water. The control group recorded the highest ammonia concentration (0.24 ± 0.02 mg L⁻¹), while the peak nitrite level was observed in treatment Z (1.87 ± 0.00 mg L⁻¹). Elevated ammonia levels resulted from the buildup of organic waste and shrimp metabolic byproducts (Saputra et al., 2024 ). Such high concentrations of ammonia pose serious risks to shrimp health. As reported by Nan et al. ( 2024 ), acute exposure can damage gill filaments and blood vessels, impairing oxygen transport. This disruption contributes to metabolic imbalances, oxidative stress, and cell death, potentially resulting in shrimp mortality. Therefore, maintaining ammonia levels within safe thresholds is essential to prevent toxic effects and support shrimp survival during the culture process (Saputra et al., 2024 ). Conclusion A gradual reduction in salinity from 10 ppt to 0.5 ppt had no significant effect on the growth of whiteleg shrimp (Litopenaeus vannamei) post-larvae (P > 0.05). However, survival rates were notably impacted. The highest survival was recorded in treatment Y—where salinity was decreased by 2 ppt every 48 hours—reaching 89.0 ± 4.0%, which was significantly higher than the control group’s survival rate of 24.0 ± 6.0% (P < 0.05). The stepwise salinity decrease also did not significantly influence hepatopancreatic tubule structure, lipid reserves, or the gut-to-muscle ratio, suggesting that the acclimatization from 10 ppt to 0.5 ppt did not compromise shrimp physiological health. Based on these findings, a salinity reduction strategy for post-larva − 10 (PL-10) vannamei shrimp in tropical regions—from 10 ppt to 0.5 ppt—can be safely implemented by lowering salinity by 2 ppt every 24 to 60 hours. Declarations Data availability The data that support the results of this study are available from the corresponding author on reasonable request. Conflict of interest The authors declare that they have no known financial conflicts of interest or personal relationships that could have appeared to influence the work reported in this paper. Author's contribution Herno Minjoyo designed the research and wrote the manuscript. Suryadi Saputra conducted the experiments and analyzed the data. Maya Meiyana conducted the experiments, while Dwi Handoko Putro collected the data and conducted the experiments. , Selfester Basi Dhoe conducted an experiment Arief Rahman Rivaie conducted the experiment. Suci Antoro conducted the data analysis , Betutu Senggagau conducted the data analysis , and Limin Santoso was responsible for the data analysis. Ethics statement All experimental procedures complied with ARRIVE guidelines and were carried out in accordance with UK legislation under the Animals Legislation Amendment (Scientific Procedures) Regulations 1986 (SI 2012/3039) and related guidelines, as well as European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. This study also complied with guidelines established by the Animal Welfare and Research Ethics Committee at Hasanuddin University, Indonesia. Thank-you note We would like to express our sincere gratitude to the Head of MS Company and the dedicated staff at the Satmakura Aquaculture Station in Campang Raya, Bandar Lampung, for their invaluable support in providing facilities, infrastructure, and personnel throughout the research activities. We also extend heartfelt thanks to the Laboratory Analysts at Lampung State University for their expert assistance in the analysis of shrimp samples. 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02:51:20","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-7191405/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7191405/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87458086,"identity":"6147c240-73ca-430f-9430-a9b11dbe481d","added_by":"auto","created_at":"2025-07-24 05:10:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":763430,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7191405/v1/248ae7f8-64f8-4b7b-ae7a-227320e06b82.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eEvaluation of gradual acclimatization of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eLitopenaeus vannamei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e post-larvae to freshwater in tropical regions: Emphasis on biological performance and physiological health responses\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e, commonly known as vannamei or whiteleg shrimp, has become one of Indonesia\u0026rsquo;s most prominent aquaculture commodities. Since its introduction in 2001, this species has played a pivotal role in revitalizing the national shrimp industry and contributing to the diversification of fishery production (Hendarajat et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Nababan et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sahabudin et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Rising demand both domestically and internationally has driven farmers to adopt more intensive farming methods, typically involving high stocking densities (Supono \u0026amp; Wardiyanto, 2008; Bagus, 2021; Cahyanurani \u0026amp; Hariri, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cahyanurani \u0026amp; Dowansiba, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cahyanurani \u0026amp; Edy, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hidayat et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Globally, \u003cem\u003eP. vannamei\u003c/em\u003e is extensively cultivated across Central and South America, the United States, East and Southeast Asia, the Middle East, and parts of Africa (Benzie, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the United States, shrimp farming traditionally associated with marine or brackish environments has expanded inland, often just a few kilometers from the coast, utilizing both saltwater and freshwater sources. This shift marks a rapidly emerging sector within the aquaculture industry (Ednoff, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). The growth of inland shrimp farming in low-salinity\u0026mdash;sometimes nearly freshwater\u0026mdash;conditions is driven by factors such as the rising cost of coastal property, environmental limitations, and increasing domestic demand for shrimp (Hopkins et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). Pacific white shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) have demonstrated the ability to thrive in freshwater systems, with successful cultivation reported at total dissolved solids (TDS) levels as low as 650 ppm at the Harbor Branch Oceanographic Institution (Van Wyk et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Van Wyk, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Similar success has been achieved in low-salinity environments throughout other parts of the U.S. (Samocha et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Ednoff, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Samocha et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIntensive shrimp farming systems, often characterized by limited environmental capacity, high stocking densities, overfeeding, and poor water quality management, can disrupt the ecological balance and increase shrimp susceptibility to disease (Zafar et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Widodo et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). One innovative approach to mitigate disease outbreaks is the implementation of low-salinity culture systems (Ariadi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ariadi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Panakorn, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Among the major pathogens in \u003cem\u003eLitopenaeus vannamei aquaculture are Vibrio species\u003c/em\u003e, particularly \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, the causative agent of Acute Hepatopancreatic Necrosis Disease (AHPND). Monitoring \u003cem\u003eVibrio populations\u003c/em\u003e is considered a key indicator of shrimp health and culture conditions (Bauer et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Pena et al., 2001). Schofield et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) showed that \u003cem\u003eV. parahaemolyticus\u003c/em\u003e AHPND maintained its pathogenicity across a wide range of salinities but caused less mortality at lower salinities. As a halophilic bacterium, \u003cem\u003eV. parahaemolyticus\u003c/em\u003e reproduces more effectively in high-salinity environments, producing \u003cem\u003ehigher levels of the pyrAB toxin\u003c/em\u003e under these conditions.\u003c/p\u003e\u003cp\u003eEstrada-P\u0026eacute;rez et al. (2020) investigated the performance of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e during AHPND outbreaks across seven shrimp pond production cycles in Mexico between 2013 and 2016. As a euryhaline species, \u003cem\u003eL. vannamei\u003c/em\u003e can adapt to a broad salinity range\u0026mdash;from 2 to 40 ppt (Scabra et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u0026mdash;making it highly suitable for inland and low-salinity aquaculture systems (Maghfiroh et al., 2019; Tahe \u0026amp; Nawang, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Low-salinity shrimp farming has been widely implemented globally (Roy et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), including in Indonesia, where it is practiced intensively in regions such as Lampung, Purworejo, Tuban, Lamongan, and Probolinggo (Ariadi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These systems typically maintain salinity levels below 10 g/L (Boyd \u0026amp; Thunjai, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The benefits of low-salinity aquaculture include reduced disease incidence, lower toxicity from harmful gases, and inhibition of pathogenic microorganisms (Samocha et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Supono, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Valencia-Casta\u0026ntilde;eda et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). From both ecological and economic perspectives, the development of low-salinity \u003cem\u003evannamei\u003c/em\u003e shrimp farming holds strong potential for sustainable aquaculture expansion (Ariadi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePost-larval \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e often experience significant stress and mortality when transferred from acclimation tanks to freshwater or low-salinity solutions with markedly different ionic compositions. Such mortality, frequently occurring within 24 hours of transfer, is commonly attributed to excessively rapid acclimation\u0026mdash;particularly when salinity decreases by more than 50% within an 8-hour window (Van Wyk et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Additional contributing factors include ionic imbalances in the destination water (McGraw \u0026amp; Scarpa, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and the lack of an appropriate transitional phase during the transfer process.\u003c/p\u003e\u003cp\u003eCurrently, there is limited information regarding the minimum ionic requirements necessary for marine shrimp, such as \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e, to survive in freshwater environments containing less than 1000 ppm total dissolved solids (TDS), as well as a lack of standardized protocols for safe freshwater acclimatization (Van Wyk et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Anonymous, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; McGraw \u0026amp; Scarpa, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; McGraw et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; McGraw \u0026amp; Scarpa, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). A more comprehensive understanding of the factors that affect shrimp survival during salinity transitions\u0026mdash;particularly the acclimation rate\u0026mdash;is essential to support the sustainable development of freshwater-based \u003cem\u003eL. vannamei\u003c/em\u003e aquaculture. This study aimed to evaluate the biological performance and physiological health of vannamei shrimp subjected to salinity reductions from seawater or high-salinity conditions to freshwater in tropical environments.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003eTest animals\u003c/p\u003e\u003cp\u003eThe test animals in this study were \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e post-larvae (PL-10), initially maintained at a salinity of 10 ppt and a temperature range of 30\u0026ndash;31.5\u0026deg;C at the hatchery. A total of 1,200 shrimp were sourced from a commercial hatchery located at Kalianda Resort, South Lampung, Indonesia. The shrimp were transported in sealed containers to the research facility, with a transit time of approximately one hour.\u003c/p\u003e\u003cp\u003eResearch procedures, data collection and research design\u003c/p\u003e\u003cp\u003eThis research was carried out at a freshwater \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e shrimp cultivation facility located in Satmakura Campang, Bandar Lampung, Indonesia. The experimental setup consisted of 12 aquaria, each with a total capacity of 120 liters and filled with 100 liters of water and was stocked with 100 shrimps every container. Each aquarium was equipped with an aeration system and a recirculating water filtration unit to ensure optimal water quality throughout the study.\u003c/p\u003e\u003cp\u003eThe materials used in this study included \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e post-larvae (PL-10) maintained at a salinity of 10 ppt, commercial shrimp feed containing 45\u0026ndash;46% protein, freshwater, seawater, and mineral supplements. At the start of the experiment, the salinity of the culture medium was adjusted to match the hatchery conditions (10 ppt). Throughout the study, the shrimp were fed artificial feed with a protein content of 45\u0026ndash;46%, administered four times daily at ad libitum levels.\u003c/p\u003e\u003cp\u003eAcclimatization to lower salinity levels (desalination) was carried out gradually until the target salinity of 0.5 ppt was achieved. This was done by periodically removing water from the containers and replacing it with freshwater, with the volume calculated to reach the intended reduction rate. This method aligns with the findings of McGraw \u0026amp; Scarpa (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), who reported improved survival rates when the acclimatization period exceeded 72 hours.\u003c/p\u003e\u003cp\u003eThe experiment employed a Completely Randomized Design (CRD) consisting of four desalination treatments, each with three replicates: (1) Control (K) \u0026mdash; a drastic reduction from 10 ppt to 0.5 ppt; (2) Treatment X \u0026mdash; gradual reduction of 2 ppt every 24 hours; (3) Treatment Y \u0026mdash; gradual reduction of 2 ppt every 48 hours; and (4) Treatment Z \u0026mdash; gradual reduction of 2 ppt every 60 hours.\u003c/p\u003e\u003cp\u003eObservations of growth and survival performance in post-larval whiteleg shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) were carried out during the desalination acclimatization process, which involved a gradual reduction in salinity from 10 ppt to 0.5 ppt. In addition to these performance metrics, physiological health parameters were assessed, including hepatopancreas condition, lipid content, and the gut muscle ratio (GMR). These evaluations were conducted on 10% of the shrimp sampled from each replicate. The percentage of hepatopancreas tubules, lipid content, and GMR were measured as indicators of internal physiological health.\u003c/p\u003e\u003cp\u003eMicrobiological analyses were also conducted, including total bacterial counts (TBC) in the culture water and total \u003cem\u003eVibrio\u003c/em\u003e counts (TVC) in both the water and shrimp samples. These measurements were taken from each replicate to assess the microbial load of the culture environment and the potential presence of pathogenic bacteria.\u003c/p\u003e\u003cp\u003eWater quality parameters\u0026mdash;including dissolved oxygen (DO), temperature, pH, salinity, conductivity, and total dissolved solids (TDS)\u0026mdash;were measured daily in the morning using a multiparameter water quality meter (Merck AZ Instrument, Model 86031). Alkalinity was analyzed according to standard methods prescribed by the American Public Health Association (APHA, 2017). Ammonia and nitrite concentrations were quantified using a UV/VIS spectrophotometer (Perkin Elmer Lambda 35), following the Indonesian National Standards: SNI 06-6989.30-2005 for ammonia and SNI 06-6989.9-2004 for nitrite. To maintain optimal environmental conditions, mineral analysis was also performed. The concentrations of magnesium (Mg\u0026sup2;⁺), potassium (K⁺), and calcium (Ca\u0026sup2;⁺) were determined using an atomic absorption spectrophotometer (AAS), based on the ASTM E663-86 standard and the operational procedures of the Shimadzu AA-7000.\u003c/p\u003e\u003cp\u003ePhysiological response parameters\u003c/p\u003e\u003cp\u003eTotal hemocyte count (THC) analysis\u003c/p\u003e\u003cp\u003eStress levels in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e were evaluated by analyzing hemocyte samples collected on day 0 (baseline) and at the end of the study. The Total Hemocyte Count (THC) was measured following the method described by Blaxhall and Daisley (1973). All analyses were conducted at the Laboratory of Lampung University.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eData analysis\u003c/h2\u003e\u003cp\u003eData on THC concentration, survival rate, specific daily growth rate, Total Vibrio Count (TVC), Total Bacterial Count (TBC), and the TVC/TBC ratio were analyzed using one-way ANOVA at a 95% confidence level. When significant differences were detected among treatments, a Least Significant Difference (LSD) post hoc test was conducted to identify specific group differences. All statistical analyses were performed using SPSS version 28. In contrast, water quality parameters were analyzed descriptively.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSurvival rate\u003c/p\u003e\u003cp\u003eThe survival rate of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e exposed to a gradual reduction in salinity from 10 ppt to 0.5 ppt varied significantly across treatments. In the control group (K), which experienced an abrupt salinity drop from 10 ppt to 0.5 ppt, the survival rate was notably low at 24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0%. In contrast, the gradual acclimatization treatments demonstrated markedly higher survival rates: 83.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5% in treatment X (2 ppt reduction every 24 hours), 89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0% in treatment Y (2 ppt reduction every 48 hours), and 85.0\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0% in treatment Z (2 ppt reduction every 60 hours). One-way ANOVA revealed that the survival rates in treatments X, Y, and Z were significantly higher than that of the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eGrowth rate\u003c/p\u003e\u003cp\u003eFrom a growth perspective, there were no statistically significant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in absolute weight gain or length increase among all treatments, including the control group. Similarly, the specific daily growth rate (SGR) showed no significant variation across treatments (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The lack of significant differences in growth parameters may be attributed to the relatively short duration of the experimental period.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBiological parameters of vannamei shrimp after salinity reduction process\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK (control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLife sustainability (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85.0\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDaily growth rate (%/day)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1 0.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9 3 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 5.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.69 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNote: Different superscripts in the same row indicate significant differences between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); K\u0026thinsp;=\u0026thinsp;control ( drastic decrease in salinity from 10 ppt to 0.5 ppt); X\u0026thinsp;=\u0026thinsp;decrease of 2 ppt every 24 hours; Y\u0026thinsp;=\u0026thinsp;decrease of 2 ppt every 48 hours; Z\u0026thinsp;=\u0026thinsp;decrease of 2 ppt every 60 hours\u003c/p\u003e\u003cp\u003ePhysiological response\u003c/p\u003e\u003cp\u003eObservations of the Total Hemocyte Count (THC) in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e post-larvae during salinity reduction from 10 ppt to 0.5 ppt revealed distinct physiological responses across treatments. In the control group, THC declined from the initial baseline of 75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u0026times; 10⁵ cells/mL to 63\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026times; 10⁵ cells/mL. Treatment X showed a further decrease to 58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 \u0026times; 10⁵ cells/mL, while treatment Z recorded a slightly higher value of 62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 \u0026times; 10⁵ cells/mL. Notably, treatment Y exhibited an increase in THC to 77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u0026times; 10⁵ cells/mL, surpassing the baseline level.\u003c/p\u003e\u003cp\u003eStatistical analysis using one-way ANOVA indicated a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between treatment Y and the other groups (control, X, and Z), suggesting that the gradual salinity reduction protocol of 2 ppt every 48 hours (treatment Y) may offer the most favorable conditions for maintaining hemocyte stability (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePhysiological response of \u003cem\u003eLitopenaeus vannamei l\u003c/em\u003earvae after salinity reduction process\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eK (control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial THC body (x 10 \u003csup\u003e5\u003c/sup\u003e cells /mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFinal THC body (x 10 \u003csup\u003e5\u003c/sup\u003e cells /mL)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e63\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e62.5\u0026thinsp;\u0026plusmn;\u0026thinsp;5.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Different superscripts in the same row indicate significant differences between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); THC\u0026thinsp;=\u0026thinsp;Total Hemocyte Count; K\u0026thinsp;=\u0026thinsp;control by drastically reducing salinity from 10 ppt directly to 0.5 ppt); X\u0026thinsp;=\u0026thinsp;decrease of 2 ppt every 24 hours; Y\u0026thinsp;=\u0026thinsp;decrease of 2 ppt every 48 hours; Z\u0026thinsp;=\u0026thinsp;with decrease of 2 ppt every 60 hours\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003cp\u003eShrimp health condition\u003c/p\u003e\n\u003cp\u003eThe outcomes of gradually reducing salinity from 10 ppt to 0.5 ppt in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. There were no significant differences (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) between the treatment groups and the control in terms of hepatopancreas structure, tubule integrity, lipid content within the hepatopancreas, or the gut muscle ratio (GMR) at the end of the acclimation period. These results suggest that gradual salinity reduction does not negatively impact the internal physiological health of the shrimp.\u003c/p\u003e\u003cp\u003eFurther evaluation revealed a marked decrease in the total \u003cem\u003eVibrio\u003c/em\u003e count (TVC) in the culture water across all treatments compared to baseline levels. However, statistical analysis showed no significant differences in TVC between the treatment groups and the control at the end of the study (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, the total bacterial count (TBC) in the water exhibited a significant reduction (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the treatment groups, particularly in treatments Y and Z, which demonstrated a substantially lower microbial load than the control.\u003c/p\u003e\u003cp\u003eAlthough both \u003cem\u003eVibrio\u003c/em\u003e and total bacterial counts declined over the course of the study, the final ratio of \u003cem\u003eVibrio\u003c/em\u003e to total bacteria did not show significant differences among the treatment and control groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These results suggest that a gradual reduction in salinity may effectively suppress the overall bacterial population, potentially enhancing water quality and promoting better shrimp health.\u003c/p\u003e\u003cp\u003eAt the end of the study, assessments of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e tissue revealed a significant decrease in both \u003cem\u003eVibrio\u003c/em\u003e and total bacterial counts across all treatments and the control. However, statistical analysis indicated no significant differences in total bacterial load between the treatments and the control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In contrast, the \u003cem\u003eVibrio\u003c/em\u003e count within shrimp tissue was significantly lower in treatment X compared to the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while treatments Y and Z did not show statistically significant differences from the control (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eAdditionally, the ratio of \u003cem\u003eVibrio\u003c/em\u003e to total bacteria in shrimp tissue declined across all treatments and the control group (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The most substantial reduction was observed in treatment X, where the \u003cem\u003eVibrio\u003c/em\u003e-to-total bacteria ratio was significantly lower than that of the control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although treatments Y and Z also demonstrated lower ratios compared to the control, the differences were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results indicate that while gradual salinity reduction can effectively lower both overall bacterial levels and \u003cem\u003eVibrio\u003c/em\u003e counts in shrimp tissue, treatment X may be particularly effective in suppressing potentially pathogenic \u003cem\u003eVibrio\u003c/em\u003e populations during the acclimatization to freshwater.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHealth parameters, TVC and TBC of vannamei shrimp after salinity reduction from 10 to 0.5 ppt\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBeginning\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehepatopancreatic tubules ( %)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e72.55\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e84.13\u0026thinsp;\u0026plusmn;\u0026thinsp;8.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e74.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.1 1 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85.12\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e83.15\u0026thinsp;\u0026plusmn;\u0026thinsp;5.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehepatopancreatic lipid (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e73.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e81.08\u0026thinsp;\u0026plusmn;\u0026thinsp;6.33 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e83.55\u0026thinsp;\u0026plusmn;\u0026thinsp;5.55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85.53\u0026thinsp;\u0026plusmn;\u0026thinsp;6.15\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e82.35\u0026thinsp;\u0026plusmn;\u0026thinsp;10.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGMR (Intestinal muscle ratio)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTVC Water ( CFU mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e26.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.81 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAir TB (CFU mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e368.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e324.5\u0026thinsp;\u0026plusmn;\u0026thinsp;29.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e410.5\u0026thinsp;\u0026plusmn;\u0026thinsp;50.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e550.0\u0026thinsp;\u0026plusmn;\u0026thinsp;60.0\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e789.0\u0026thinsp;\u0026plusmn;\u0026thinsp;109.0\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTVC/TBC Ratio of Water (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.98 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBody TB (CFU mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2240.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e967.5\u0026thinsp;\u0026plusmn;\u0026thinsp;90.5ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e816.0\u0026thinsp;\u0026plusmn;\u0026thinsp;8.0ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e906.5\u0026thinsp;\u0026plusmn;\u0026thinsp;261.5ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e855.5\u0026thinsp;\u0026plusmn;\u0026thinsp;121.5ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTVC Body (CFU mL \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e288.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9.33\u0026thinsp;\u0026plusmn;\u0026thinsp;15.31ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.33\u0026thinsp;\u0026plusmn;\u0026thinsp;14.50ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5.0\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6.50ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTVC/TBC Body Ratio (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.90\u0026thinsp;\u0026plusmn;\u0026thinsp;2.29ᵇ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05ᵃ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97ᵃ\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"6\"\u003eNote: Different superscripts in the same row indicate significant differences between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); TVC\u0026thinsp;=\u0026thinsp;Total Vibrio Count; TBC\u0026thinsp;=\u0026thinsp;Total Bacterial Count; K\u0026thinsp;=\u0026thinsp;control ( drastically decreasing salinity from 10 ppt to 0.5 ppt); X\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 24 hours; Y\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 48 hours; Z\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 60 hours\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWater quality parameters\u003c/p\u003e\u003cp\u003eThroughout the study, salinity was gradually reduced from 10 ppt to 0.5 ppt during the rearing of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e, with careful monitoring of water quality parameters. Overall, the recorded parameters\u0026mdash;particularly temperature, dissolved oxygen (DO), pH, and alkalinity\u0026mdash;remained within acceptable ranges to support shrimp survival and health (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). However, nitrite levels were consistently elevated across all treatments, potentially posing a health risk. In addition, concentrations of key minerals such as calcium, magnesium, and potassium were relatively low, which could negatively impact osmoregulatory function and general physiological performance.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eWater quality and mineral parameters after salinity reduction from 10 to 0.5 ppt\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBeginning\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c6\" namest=\"c3\"\u003e\u003cp\u003eTreatment\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eK\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eY\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZ\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e7.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTemperature ( \u003csup\u003e0\u003c/sup\u003e C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e30.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e31.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDO ( mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAlkalinity ( mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e143.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e58.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e54.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.0 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e51.5\u0026thinsp;\u0026plusmn;\u0026thinsp;4.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNitrite ( mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAmmonium ( mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConductivity (\u0026micro;S cm \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e39.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1,129\u0026thinsp;\u0026plusmn;\u0026thinsp;160.5 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1081.67\u0026thinsp;\u0026plusmn;\u0026thinsp;255 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e975\u0026thinsp;\u0026plusmn;\u0026thinsp;76 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1,059.3\u0026thinsp;\u0026plusmn;\u0026thinsp;173 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTDS (mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e19.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e564.0\u0026thinsp;\u0026plusmn;\u0026thinsp;81 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e613.67\u0026thinsp;\u0026plusmn;\u0026thinsp;180 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e487.33\u0026thinsp;\u0026plusmn;\u0026thinsp;37 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e529.25\u0026thinsp;\u0026plusmn;\u0026thinsp;55 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCalcium (mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e76.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMagnesium (mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e102.9\u0026thinsp;\u0026plusmn;\u0026thinsp;15.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePotassium (mg L \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e28.34\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eNote: Different superscripts in the same row indicate significant differences between treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); K\u0026thinsp;=\u0026thinsp;control (drastically decreasing salinity from 10 ppt to 0.5 ppt); X\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 24 hours; Y\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 48 hours; Z\u0026thinsp;=\u0026thinsp;decreasing salinity by 2 ppt every 60 hours\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed the effects of gradual salinity reduction on the survival of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e post-larvae (PL-10), using a reduction rate of 2 ppt implemented at three different time intervals, along with a control group that experienced an abrupt drop in salinity from 10 ppt to 0.5 ppt. The results revealed significant differences in survival rates between the control and all gradual acclimation treatments (X: 24-hour interval; Y: 48-hour interval; Z: 60-hour interval). The survival rates observed in this study were notably higher than those reported by McGraw et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), who recorded only 5% and 13% survival at 48 and 24 hours, respectively, for PL-10 shrimp acclimated to 1 ppt. In comparison, this study achieved survival rates of 83.7\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5% (24 h), 89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0% (48 h), and 85.0\u0026thinsp;\u0026plusmn;\u0026thinsp;22.0% (60 h), aligning more closely with the survival performance typically observed in older post-larvae (PL-15\u0026ndash;PL-20) in previous research.\u003c/p\u003e\u003cp\u003eWilliam et al. (2004) also reported comparable survival outcomes in PL15 shrimp acclimated to low salinity with added mineral ions. Notably, the survival rates observed in the present study were slightly higher than those documented by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who conducted acclimation at a final salinity of 0.5 ppt, and Abrori et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who used 5 ppt. McGraw and Scarpa (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) found that acclimation duration and habituation period did not significantly impact 24-hour shrimp survival, which ranged from 76\u0026ndash;81% and 73\u0026ndash;82%, respectively. However, other studies indicate that gradual acclimation enhances survival outcomes. Jayasankar et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) demonstrated near-total survival of \u003cem\u003eP. vannamei\u003c/em\u003e acclimated to 5 ppt through a stepwise salinity reduction protocol, while McGraw and Scarpa (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) further showed improved survival at 1 ppt when the acclimation period was extended to 72 hours.\u003c/p\u003e\u003cp\u003eThe osmoregulatory capacity of crustaceans serves as a reliable indicator of their physiological condition and acts as a sensitive biomarker for evaluating the effects of environmental stressors, pollutants, or pathogens (Lignot et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). These organisms are capable of maintaining internal osmotic equilibrium, and fluctuations in this ability may signal physiological stress.\u003c/p\u003e\u003cp\u003eRegarding growth performance, no significant differences were observed between the treatment groups and the control. Nonetheless, treatment Z exhibited a marginally higher daily growth rate compared to the other treatments. Overall, the growth outcomes in this study were superior to those reported by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), suggesting that the salinity reduction protocol applied\u0026mdash;particularly in treatment Y\u0026mdash;closely approached an optimal acclimatization strategy. This likely reflects a physiological equilibrium between the shrimp\u0026rsquo;s internal regulatory systems and their external environment. These findings align with Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who similarly reported that gradual salinity reduction did not significantly impact growth.\u003c/p\u003e\u003cp\u003eFrom a physiological standpoint, the total hemocyte count (THC) in \u003cem\u003eL. vannamei\u003c/em\u003e PL-10 declined following salinity reduction from 10 ppt to 0.5 ppt relative to initial values. Nonetheless, statistical analysis revealed no significant differences in THC across treatments and the control, with the exception of treatment Y (77.5\u0026thinsp;\u0026plusmn;\u0026thinsp;8.5 \u0026times; 10⁵ cells/mL), which also yielded the highest survival rate (89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0%). These findings suggest that the carefully managed salinity reduction protocol implemented in treatment Y contributes to maintaining physiological homeostasis and enhancing survival in whiteleg shrimp post-larvae.\u003c/p\u003e\u003cp\u003ePhysiological responses of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e to reduced salinity during acclimatization were assessed by examining hepatopancreatic tubule structure, lipid content within the hepatopancreas, and the gut muscle ratio (GMR) before and after salinity reduction. The analysis revealed no statistically significant differences between the treatment groups and the control. These findings are consistent with those reported by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe hepatopancreas is a critical organ in shrimp, essential for digestion and metabolic regulation (Wang et al., 2023). The consistent stability of this organ across all treatments suggests that the gradual acclimation protocol successfully minimized osmoregulatory stress, thereby preserving its functional integrity. This maintenance of hepatopancreatic function is crucial for supporting efficient metabolic and digestive processes during periods of salinity reduction. Huang et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) emphasized the role of lipids\u0026mdash;particularly triglycerides and phospholipids\u0026mdash;in shrimp osmoregulation, noting their function as key energy reserves and their importance in sustaining physiological activity and cell membrane stability under changing environmental conditions.\u003c/p\u003e\u003cp\u003eIn this study, the gut muscle ratio (GMR)\u0026mdash;a key indicator of digestive efficiency and muscle development\u0026mdash;remained consistent across all treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As highlighted by Peregrino (2006), the relative proportion of muscle to gut serves as a reliable metric for evaluating postlarval quality. These results suggest that gradual salinity reduction did not negatively impact the digestive system or overall growth of \u003cem\u003eL. vannamei\u003c/em\u003e. The acclimatization from 10 ppt to 0.5 ppt was well tolerated and appears to support physiological resilience. This aligns with findings from Lin and Chen (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), who demonstrated the species\u0026rsquo; robust osmoregulatory capacity across a broad salinity range, enabling metabolic equilibrium and sustained health under salinity stress. Similar observations were also reported by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), further reinforcing the effectiveness of gradual salinity adjustment.\u003c/p\u003e\u003cp\u003eOne of the primary contributors to disease outbreaks in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e aquaculture in Indonesia is \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, a pathogenic bacterium known for its rapid proliferation in high-salinity conditions, including brackish water systems. In this study, observations\u0026mdash;particularly at the conclusion of the trial period\u0026mdash;revealed a substantial reduction in total bacterial count (TBC) within both the rearing water and the shrimp, relative to initial measurements. This decline was accompanied by a marked decrease in total \u003cem\u003eVibrio\u003c/em\u003e count (TVC) in both the culture water and shrimp samples.\u003c/p\u003e\u003cp\u003eSimilar trends were reported by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who also documented a reduction in total \u003cem\u003eVibrio\u003c/em\u003e count (TVC) in shrimp tissue across various salinity treatments, although inter-treatment variability was minimal. Conversely, total bacterial count (TBC) in the culture water increased in all treatment groups\u0026mdash;except the control\u0026mdash;by the end of the study period. This rise is likely attributable to the accumulation of organic matter, including uneaten feed, metabolic byproducts, and shrimp fecal matter, as previously highlighted by Seethalakshmi et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the increase in total bacterial count (TBC) in the culture water, the ratio of total \u003cem\u003eVibrio\u003c/em\u003e count (TVC) to TBC in both the culture medium and shrimp tissue declined significantly, serving as a positive indicator of shrimp health. Gradual and carefully controlled salinity reduction appears to play a vital role in maintaining physiological homeostasis in \u003cem\u003eL. vannamei\u003c/em\u003e (Lin \u0026amp; Chen, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), thereby reducing vulnerability to microbial infections. Effective acclimatization also supports immune system stability during environmental transitions (Chang et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). As salinity approached freshwater levels, the prevalence of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e\u0026mdash;which thrives under higher salinity conditions\u0026mdash;was notably suppressed. These findings are consistent with Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who reported that gradual, non-abrupt salinity adjustments reduce physiological stress and mitigate the risk of pathogenic bacterial infections, particularly those caused by \u003cem\u003eVibrio\u003c/em\u003e spp.\u003c/p\u003e\u003cp\u003eAnalysis of total \u003cem\u003eVibrio\u003c/em\u003e count (TVC) and total bacterial count (TBC) in the bodies of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e at the conclusion of the acclimation period revealed a significant reduction in both bacterial populations. This decline underscores the positive impact of a carefully managed, gradual salinity reduction in promoting shrimp health by limiting the prevalence of pathogenic bacteria such as \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, thereby lowering the risk of infection and disease. While TBC decreased significantly across all groups\u0026mdash;including the control\u0026mdash;no statistically significant differences were detected among treatments. In contrast, TVC exhibited a significant decline, particularly in treatment X, which also displayed a notable change in the TVC/TBC ratio within shrimp tissue. Although these results differ slightly from those documented by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), they are in alignment with the broader conclusions presented by Chang et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which emphasize the effectiveness of controlled acclimation protocols in mitigating microbial threats.\u003c/p\u003e\u003cp\u003eSimultaneously, concentrations of key minerals\u0026mdash;magnesium (Mg\u0026sup2;⁺), calcium (Ca\u0026sup2;⁺), and potassium (K⁺)\u0026mdash;declined significantly following salinity reduction to 0.5 ppt (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This decrease is likely linked to the increased utilization of these ions by shrimp to support critical physiological functions, such as osmoregulation and exoskeleton development. Magnesium plays a central role in maintaining ionic equilibrium and is essential for exoskeletal structure (Davis et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). As demonstrated by Cheng et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), shrimp actively extract minerals from their environment to counter osmotic challenges in low-salinity conditions. Potassium, vital for sustaining intracellular ion gradients, osmotic pressure, and neuromuscular activity, is similarly absorbed at elevated rates during hypoosmotic stress (Roy et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Huong et al., 2010). Calcium is another essential element, particularly important for molting, and its decreased levels reflect increased physiological demand during acclimation\u0026mdash;a trend consistent with observations by Davis et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFluctuations in mineral concentrations within aquaculture systems play a vital role in sustaining the ecological balance of the culture environment. These minerals are not only critical for the physiological well-being of shrimp but also serve as essential nutrients for the growth and metabolism of aquatic microorganisms (Zhang et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The decline in mineral levels observed during salinity acclimatization is influenced both by increased mineral uptake by shrimp and the biological activity of resident microbial communities. To counter these reductions and maintain optimal shrimp performance, mineral supplementation\u0026mdash;specifically with magnesium, calcium, and potassium\u0026mdash;is essential throughout the salinity reduction process. Previous studies have demonstrated that such supplementation enhances shrimp survival and growth during transitions to low-salinity conditions (Saputra et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Wang et al. (2023) further emphasized that targeted mineral enrichment improves osmoregulatory capacity, thereby promoting metabolic stability and facilitating successful adaptation under hypoosmotic stress.\u003c/p\u003e\u003cp\u003eDuring this study, the gradual reduction of salinity from 10 ppt to 0.5 ppt was accompanied by measurable shifts in water quality parameters (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Despite these changes, dissolved oxygen (DO) levels consistently remained within the optimal range, ensuring sufficient support for the physiological requirements and post-larval development of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. The observed DO values satisfied the oxygen demands necessary for aerobic respiration, a critical component of growth and molting processes. Water temperature across all treatment groups also remained within the optimal spectrum, ranging from 30.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u0026deg;C to 31.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u0026deg;C. These findings align with the work of Venkateswarlu et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), who identified 30\u0026deg;C as the ideal thermal condition for \u003cem\u003eL. vannamei\u003c/em\u003e. Together, stable temperatures and adequate DO levels fostered a conducive environment for metabolic function and physiological stability throughout the acclimation period.\u003c/p\u003e\u003cp\u003eThe application of a consistent and accurately managed salinity reduction protocol\u0026mdash;targeting a final salinity of 0.5 ppt\u0026mdash;proved effective in alleviating osmotic stress in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. Maintaining stable salinity conditions is critical for minimizing the risk of osmotic shock, which can significantly compromise shrimp health and survival during transitions from high-salinity to freshwater environments. Chen et al. (2015) demonstrated that a gradual acclimatization strategy enables post-larval shrimp to adjust more efficiently to salinity shifts, thereby mitigating the physiological stress linked to osmoregulatory demands.\u003c/p\u003e\u003cp\u003eFollowing the salinity reduction, conductivity and total dissolved solids (TDS) levels declined significantly across all treatment groups (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These changes reflect a reduced concentration of dissolved ions and particulates in the culture water. Conductivity values were notably lower in all treatments compared to the control, indicating diminished ion availability. Adequate levels of dissolved ions are essential for efficient osmoregulation in \u003cem\u003eL. vannamei\u003c/em\u003e, particularly under conditions of fluctuating salinity (Duan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The observed decline in conductivity and TDS suggests that the shrimp were able to physiologically adapt to low-salinity conditions without disruption to metabolic function, despite shifts in water chemistry. These results are consistent with findings by Saputra et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), who emphasized the value of gradual mineral and salinity adjustments in supporting shrimp adaptation during environmental transitions.\u003c/p\u003e\u003cp\u003eThe implementation of a controlled and consistent salinity reduction protocol\u0026mdash;reaching a final concentration of 0.5 ppt\u0026mdash;effectively mitigated osmotic stress in \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. Maintaining stable salinity levels is essential in preventing osmotic shock, which can severely impact shrimp health and survival during transitions from saline to freshwater environments. Supporting these findings, Chen et al. (2015) demonstrated that gradual acclimatization enhances the ability of post-larval shrimp to cope with salinity fluctuations, thereby reducing the physiological burden associated with osmoregulatory adjustment.\u003c/p\u003e\u003cp\u003eAlkalinity was a key water quality parameter evaluated in this study, and results indicated that levels remained relatively stable across all treatment groups. This consistency suggests that the shrimp experienced minimal acid-base disruption throughout the acclimatization process. Stable alkalinity is essential for preserving acid-base homeostasis, which underpins effective osmoregulatory function and nitrogen excretion\u0026mdash;two critical physiological processes influencing the survival of \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e during cultivation (Zhang et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Saputra et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFluctuations in ammonia and nitrite concentrations throughout the study reflected ongoing biological processes within the culture water. The control group recorded the highest ammonia concentration (0.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 mg L⁻\u0026sup1;), while the peak nitrite level was observed in treatment Z (1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 mg L⁻\u0026sup1;). Elevated ammonia levels resulted from the buildup of organic waste and shrimp metabolic byproducts (Saputra et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Such high concentrations of ammonia pose serious risks to shrimp health. As reported by Nan et al. (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), acute exposure can damage gill filaments and blood vessels, impairing oxygen transport. This disruption contributes to metabolic imbalances, oxidative stress, and cell death, potentially resulting in shrimp mortality. Therefore, maintaining ammonia levels within safe thresholds is essential to prevent toxic effects and support shrimp survival during the culture process (Saputra et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA gradual reduction in salinity from 10 ppt to 0.5 ppt had no significant effect on the growth of whiteleg shrimp (Litopenaeus vannamei) post-larvae (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). However, survival rates were notably impacted. The highest survival was recorded in treatment Y\u0026mdash;where salinity was decreased by 2 ppt every 48 hours\u0026mdash;reaching 89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0%, which was significantly higher than the control group\u0026rsquo;s survival rate of 24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The stepwise salinity decrease also did not significantly influence hepatopancreatic tubule structure, lipid reserves, or the gut-to-muscle ratio, suggesting that the acclimatization from 10 ppt to 0.5 ppt did not compromise shrimp physiological health. Based on these findings, a salinity reduction strategy for post-larva \u0026minus;\u0026thinsp;10 (PL-10) vannamei shrimp in tropical regions\u0026mdash;from 10 ppt to 0.5 ppt\u0026mdash;can be safely implemented by lowering salinity by 2 ppt every 24 to 60 hours.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the results of this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known financial conflicts of interest or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026apos;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHerno Minjoyo designed the research and wrote the manuscript. Suryadi Saputra conducted the experiments and analyzed the data. Maya Meiyana conducted the experiments, while Dwi Handoko Putro collected the data and conducted the experiments. , Selfester Basi Dhoe conducted an experiment Arief Rahman Rivaie conducted the experiment. Suci Antoro conducted the data analysis , Betutu Senggagau conducted the data analysis , and Limin Santoso was responsible for the data analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures complied with ARRIVE guidelines and were carried out in accordance with UK legislation under the Animals Legislation Amendment (Scientific Procedures) Regulations 1986 (SI 2012/3039) and related guidelines, as well as European Union Directive 2010/63/EU on the protection of animals used for scientific purposes. This study also complied with guidelines established by the Animal Welfare and Research Ethics Committee at Hasanuddin University, Indonesia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThank-you note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to express our sincere gratitude to the Head of MS Company and the dedicated staff at the Satmakura Aquaculture Station in Campang Raya, Bandar Lampung, for their invaluable support in providing facilities, infrastructure, and personnel throughout the research activities. We also extend heartfelt thanks to the Laboratory Analysts at Lampung State University for their expert assistance in the analysis of shrimp samples. Lastly, we deeply appreciate the contribution of the National Research and Innovation Agency (BRIN) for conducting additional sample analyses that enriched the outcomes of this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAnonymous, 2002. 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Pathogenicity of Acute Hepatopancreatic Necrosis Disease (AHPND) on the freshwater prawn, Macrobrachium rosenbergii, and Pacific White Shrimp, \u003cem\u003ePenaeus vannamei \u003c/em\u003e, at various salinities. Aquaculture Research, 52(4): 1480 1489\u003c/li\u003e\n \u003cli\u003eScabra, AR, M. Marzuki, N. Cokrowati, BDH Setyono, LF Mulyani. 2021. Increasing Solution of Calcium Through The Addition of Ketapang Leaf Terminalia Catappa In Fresh Water Medium For Vannamei Shrimp Cultivation \u003cem\u003eLitopennaeus vannamei \u003c/em\u003e. Unram Fisheries Journal, 11(1): 35\u0026ndash;49.\u003c/li\u003e\n \u003cli\u003eScabra AR, Marzuki M., Alhijrah MR, 2023 [Addition of calcium carbonate (CaCO3) and magnesium sulfate (MgSO4) to vannamei shrimp ( \u003cem\u003eLitopenaeus vannamei \u003c/em\u003e) rearing media in fresh water]. Journal of Tropical Biology 23(1):392-401. [in Indonesian]\u003c/li\u003e\n \u003cli\u003eSeethalakshmi PS, Rajeev R., Kiran GS, Selvin J., 2021 Shrimp disease management for sustainable aquaculture: innovations from nanotechnology and biotechnology. Aquaculture International 29(4):1591-1620.\u003c/li\u003e\n \u003cli\u003eSupono, Wardiyanto. 2008. Evaluation of White Shrimp Cultivation ( \u003cem\u003eLitopenaeus vannamei \u003c/em\u003e) by Increasing Stocking Density in Intensive Ponds. Seminar on Research Results \u0026amp; Community Service, University of Lampung, 237-242.\u003c/li\u003e\n \u003cli\u003eSupono, S. 2019. Low Salinity Vaname Shrimp Cultivation, Solution for Cultivation in Critical Land. Graha Ilmu, Yogyakarta\u003c/li\u003e\n \u003cli\u003eSupono, Nurdianti L., Fidyandini HP, 2023 Effect of different ratios of sodium and potassium on the growth and survival rate of Pacific white shrimp ( \u003cem\u003eLitopenaeus vannamei \u003c/em\u003e) cultured in freshwater. 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Relationship of Environmental Parameters with the Prevalence of Infectious Myonecrosis Virus in Intensive Vannamei Shrimp ( \u003cem\u003eLitopenaeus vannamei \u003c/em\u003e) Ponds in Kaur Regency. Naturalis: Journal of Natural Resources and Environmental Management Research, 10(1): 13\u0026ndash;24.\u003c/li\u003e\n \u003cli\u003eZafar, MA, MM Haque, MSB Aziz, MM Alam, 2015. Study on water and soil quality parameters of shrimp and prawn farming in the southwest region of Bangladesh. Journal of the Bangladesh Agricultural University, 13(1): 153\u0026ndash;160.\u003c/li\u003e\n \u003cli\u003eZhang Q., Yu Y., Luo Z., Li F., 2023. Hepatopancreas color as a phenotype to indicate the infection process of \u003cem\u003eVibrio parahaemolyticus \u003c/em\u003ein Pacific white shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. Aquaculture 572:739545.\u003c/li\u003e\n \u003cli\u003eZhang X., Song X., Huang J., 2016. Impact of \u003cem\u003eVibrio parahaemolyticus \u003c/em\u003eand white spot syndrome virus (WSSV) co-infection on survival of penaeid shrimp \u003cem\u003eLitopenaeus vannamei\u003c/em\u003e. Chinese Journal of Oceanology and Limnology 34(6):1278-1286. \u003c/li\u003e\n \u003cli\u003eZhang X., Zhang Y., Zhang Q., Liu P., Guo R., Jin S., Liu J., Chen L., Ma Z., Liu Y., 2020. Evaluation and analysis of water quality of marine aquaculture area. International Journal of Environmental Research and Public Health 17(4):1446. 572:739545.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"a8c27b88-d40b-4490-bfb0-39d74d25d3b3","identifier":"10.13039/501100001868","name":"National Science Council","awardNumber":"FY 2024","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"National Research and Innovation Agency Republic of Indonesia","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Litopenaeus vannamei, Survival, physiological health, freshwater","lastPublishedDoi":"10.21203/rs.3.rs-7191405/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7191405/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVannamei shrimp (\u003cem\u003eLitopenaeus vannamei\u003c/em\u003e) have long been cultivated in brackish or marine waters in Indonesia. However, production has faced challenges due to outbreaks of \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e. Research suggests that this pathogen does not thrive at very low salinity levels, prompting interest in low-salinity culture systems. To explore this approach, a study was conducted from March to April 2024 to evaluate the effects of gradual salinity reduction on the survival and physiological health of \u003cem\u003eL. vannamei\u003c/em\u003e post-larvae (PL-10). The research was carried out at PT. Mochtar Sani Corporation (MSC), Campang Raya, Bandar Lampung, Indonesia. A Completely Randomized Design (CRD) was employed, consisting of four treatment groups, each replicated three times, namely Control K (Sudden salinity reduction from 10 ppt to 0.5 ppt); Treatment X ( Gradual salinity reduction of 2 ppt every 24 hours); Treatment Y (Gradual salinity reduction of 2 ppt every 48 hours); and Treatment Z (Gradual salinity reduction of 2 ppt every 60 hours).The highest survival rate was recorded in Treatment Y (89.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.0%). Statistically, the control group exhibited a significantly lower survival rate (24.0\u0026thinsp;\u0026plusmn;\u0026thinsp;6.0%) compared to all gradual reduction treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), though no significant differences were observed among the three gradual treatments (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Additionally, the specific daily growth rate showed no significant differences among treatments, including the control group (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Physiological health indicators\u0026mdash;such as hepatopancreas condition, lipid levels, and intestinal muscle ratio\u0026mdash;remained within normal ranges throughout the acclimatization period, indicating that gradual salinity reduction did not negatively impact shrimp health.\u003c/p\u003e","manuscriptTitle":"Evaluation of gradual acclimatization of Litopenaeus vannamei post-larvae to freshwater in tropical regions: Emphasis on biological performance and physiological health responses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-24 05:02:37","doi":"10.21203/rs.3.rs-7191405/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"be81fc42-dd30-4d81-9480-5be6a1bfa643","owner":[],"postedDate":"July 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51961426,"name":"Aquaculture and Mariculture"}],"tags":[],"updatedAt":"2025-07-24T05:02:37+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-24 05:02:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7191405","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7191405","identity":"rs-7191405","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}