Growth Performance and Survivability of the Asian Seabass Lates Calcarifer Reared Under Hyper-Saline, Hypo-Saline and Freshwater Environments in a Closed Aquaculture System

Salinity is one of the most critical environmental parameters regarding sh physiology, modifying food intake and growth performance in many sh species. The present study has investigated the effects of different salinity levels on growth performance, feeding and survival of Asian seabass Lates calcarifer juveniles. Asian seabass juveniles were reared at 0, 5, 22, 36, and 42 ppt salinity. One ve hundred ninety-eight sh individuals with an average weight 1.2 ± 0.11 g were randomly distributed (166 sh/Tank) in 5 concrete tanks (30×6×4 feet) each; length × width × depth) and volume 19,122 L, for total forty days. Initially feeding rate of 6% biomass per day with 42% crude protein-containing diets daily. The feeding rate adjusted according to sh biomass every week. The results showed that salinity level had a signicant effect on the weight gain, average daily weight gain, specic growth rate, feed conversion ratio, survival rate, total biomass and health indices (p < 0.05). The highest WG (39.11±1.49 g), ADWG, (1.00±0.12 g), SGR (8.74±0.03 % d -1 ) and lowest FCR (0.96±0.20) observed with T3 (22 ppt salinity) treatment, which was signicantly higher compared to other treatment groups (p < 0.05). Among the health indices, the highest hepatosomatic index and viscerosomatic index found with 22 ppt salinity treatment, which was also signicantly higher than the other treatment groups (p < 0.05). No signicant differences were found among the treatments in terms of survival rate (p > 0.05), but the maximum survival rate (98.89±0.0 %) in T3 (22 ppt) and T2 (5 ppt). The maximum level of crude proteins (19.99±1.4%) was found in the whole-body biochemical composition (% of wet weight) of Asian sea bass juveniles in T3 treatment group reared at 22 ppt salinity. The second-order polynomial regression showed that 20 ppt salinity is optimum for the best growth of Asian sea bass. Thus, our present work would provide valuable information to the sh farmers for culturing the sea bass as well as its management along the inland and coastline of Pakistan.


Introduction
Lates calcarifer (Bloch, 1790) is commonly known as "Barramundi" or "giant sea perch" or "Asian sea bass" throughout the world. It is one of the most suitable species for aquaculture, its tolerance to broad environmental conditions, gregarious (readily school), delicately-avoured tasty, nutritious meat and fast growth rate that can be reached to 1.5 to 3.0 kg in a year when its environmental conditions are kept optimum, the maximum weight is about 60 kg (Allen et al. 2002), with sustained high demand and market price in both domestic and export markets (Hassan et al. 2020a; Rao et al. 2013). Asian sea bass is a comparatively strong ecological tolerance that tolerates a broad range of salinity from freshwater to complete seawater from 0 to 56 ppt salinity (FAO 2017). Salinity is a critical environmental factor which can highly effect on the physiology of aquatic organisms (Urbina & Glover 2015). Asian seabass has wide physiological tolerances growing in marine, brackish, and freshwater (Anil et al., 2010; Ganzon-Naretetal 2013; Venkatachalam et al. 2018; Yue et al. 2009). Salinity also has an antagonistic effect on growth, survival, fecundity, physiology and osmoregulation (Smith 2003;Smith and Schindler 2009;Anni et al. 2016). Fish at the extreme of their salinity tolerance range often exceed their osmoregulation ability (Sampaio and Bianchini 2002). The rapid rise in salinity disrupts physiological procedures in sh, leading to greater cortisol concentrations in the blood (Tsuzuki et al. 2007).
Culture of L. calcarifer is relatively new in Pakistan (Shah et al. 2020), To initiate advance aquaculture, the development of breeding, larviculture and farming technology is an urgent need to increase and establish Asian sea bass culture industry throughout the country. Speci c water parameters and seed production management are required for effective aquaculture practice to avoid the wild sheries resources declining (Shah et al. 2020) for excellent growth performance and successful aquaculture necessary optimum parameters. Therefore, the objective of this study was to investigate the growth performance, survival, and health status of Asian seabass with a wide range of salinities in a closed aquaculture system.

Experimental design
The present study was conducted at the Sindh sh hatchery near Hawkes Bay town from March 2019 to Jan 2020. A total of 598 uniform sized (1.24±1.08 grams) sea bass juveniles were collected from the coastal area of Sindh Sakro and stocked (166 sh/Tank) in ve treatments (T1, T2, T3, T4 and T5). Each treatment tank was 30×6×4 feet containing 0, 5, 22, 35 and 42 ppt salinity by following the methods of Azodi et al. (2016) with some modi cations. Tank water was regulated by blending ltered water from the ocean and freshwater. Water was exchanged for the maintenance of the water quality by up to 50% every 2 days. Fish were fed at 6% of body weight per day (BW day -1 ) for 40 days and the feeding rate adjusted every week. The extracted pellet (42.0% crude protein) was used for the manual feeding at two distinct satiation levels twice daily. After 3 hours of feeding, the extra feed was removed. The total consumption of food was reported daily in each treatment. A 42% CP-level diet was formed using various components (Table 1), referred to as the entire diet/ration structure. This diet was offered regularly based on the daily wet bodyweight of the individuals.

Feed formulation and preparation
Feed ingredients were collected from Karachi's local market and formulated as previously methods described by Ghosh et al. (2011). The major source of protein used in diets was shmeal, and the levels of protein were preserved at 42%. The feed ingredients have been combined to form a dough with sh oil and water. The palletizer was carried through the dough to create the necessary pellet diet. Under dry conditions, the feed was stored in a freezer until used. Proximate dry matter, crude fat, crude protein, crude bres, and moisture levels were determined in a percentage of the dried feed specimens (Table 1).

Samples collection and Biochemical analysis
The biochemical analysis of feed and sh carcass samples was carried out based on (AOAC 2000). At the end of the experiment, ve sh were removed from each tank and then dissected to weigh the liver and viscera, then HSI and VSI were determined. Techniques (AOAC 2000) were used to analyze crude lipid (CL), moisture and crude protein (CP). Moisture was measured at 105 o Celsius in the oven for 24 h (Labostar-LG122 Tabia Espec, Osaka, Japan). Chloroform/methanol (2:1v/v) extraction process predicted for crude lipids. (Folch et al. 1957) The CP analyzed using the automatic processing of Kjeldahl (Buchi430/) Using the Kjeltec method (N 6.25) with the automatic Kjeldahl system (Buchi 430/323) Model 1265, Moline IL, USA). Furthermore, for the determination of ash, the sample was burned at 550 C in a mu e furnace for 3 hours. Gross energy (GE) has been approximated for formulated diets factors 23.62, 39.5 and 17.56 KJ/g for CP, EE and carbohydrate respectively were used (NRC 1993).

Water quality parameters
Water quality parameters such as temperature, salinity, dissolved oxygen (DO), and pH were recorded daily using Celsius glass thermometer, Handheld Refractometer, mobile digital DO-meter (Model: HI9146) and Digital pH meter, respectively. The concentrations of ammonia, alkalinity, nitrate, and nitrite were measured by following the methods of APHA (1995). Details of the water quality parameters recorded during the study period are given in Table 2.

Growth performance analysis
The wet weight gain, average daily weight gain (ADWG), speci c growth rate (SGR), feed conversion ratio (FCR), hepatosomatic index (HSI), viscerosomatic index (VSI), Fulton's condition factor (CF), survival rate (SR) and total biomass were calculated with the following formula adopted by Hassan  All data were analyzed using STATISTICA V.13 software (StatSoft Inc., Tulsa, OK, USA). Residuals were tested for normality (Shapiro-Wilk test) and homogeneity of variance (plot of residuals versus predicted values). The percentages data were natural log (LN)-transformed before analysis. The effects of different salinity group on the growth performance and health indices of Asian seabass juveniles at different culture periods were analyzed using a repeated-measures ANOVA model containing culture period ( xed repeated factor) and different salinity groups ( xed factor) main effects as well the culture period × different salinity groups interaction term. Then, the model was decomposed into a series of one-way ANOVA was run to test the effects of different salinity levels on the wet weight gain, average daily weight gain, speci c growth rate, hepatosomatic index, viscerosomatic index, Fulton's condition factor, feed conversion ratio, survival rate and biomass for different culture periods. A posteriori analysis was performed using Tukey's multiple comparisons procedures. A signi cance level of p < 0.05 was used for all statistical tests.

Results
Physicochemical parameters Table 2 shows the minimum and maximum values of water quality parameters in ve experimental groups recorded as water pH and the temperature was range from 8.10 to 8.81 and 29.40 to 29.74°C, while dissolved oxygen (DO) was found in the range from 7.16 to 8.16 mg/L, dissolved ammonia concentration was 0.012 to 0.072; whereas, alkalinity was between 126.1 to 162.4 mg/L, nitrates from 0.004 to 2.57 mg/L and nitrates 0.010 to 0.014mg/L respectively.

Growth performance
The saturated repeated-measures ANOVA model revealed a signi cant effect on the culture period × different salinity groups interaction term (p < 0.01). Therefore, the model was decomposed into a series of a lower-order statistical model. Our results showed that salinity level had a signi cant effect on the weight gain, average daily weight gain, speci c growth rate total biomass and health indices (p < 0.05) ( Table 3). The highest weight gain (39.11±1.49 g), average daily weight gain (1.00±0.12 g), speci c growth rate (8.74±0.03% d -1 ) and production (6654±4.20 g) observed with 22 ppt salinity treatment, which was signi cantly higher compared to other treatment groups (p < 0.05) followed by salinity 42 ppt (T5), 36 ppt salinity (T4), 5 ppt and 0 ppt (T1) respectively. However, the growth curve decreased with increased salinity above 36 to 42 ppt. The second-order polynomial regression showed that 20 ppt salinity is optimum for the best growth of Asian sea bass

Morphological indices
Among the health indices, the highest hepatosomatic index and viscerosomatic index found with 22 ppt salinity treatment, which was also signi cantly higher than the other treatment groups (p < 0.05). The better condition factor also found in T3 compared to other treatments.

Cannibalism and survival
However, no signi cant differences were found among the treatments in terms of survival rate (p > 0.05), but the maximum survival rate (99.39±0.0 %) in T3 (22 ppt). The cannibalism noted in T1 and T4 treatments.

Proximate composition of sh carcass
The proximate carcass composition was calculated on basis wet weight and presented in Table 4 carcass content of moisture and protein did not signi cantly affect by salinity. Conversely, carcass content of lipid and ash signi cantly differed among the treatments, where lipid carcass of T1, T2, T3 is lower compared to T4 and T5. The moisture content and protein in carcass did not signi cantly effect by salinity, were moisture of T1 is higher than the other treatments. There was a positive correlation between the carcass content of crude proteins and increasing of salinity level, The maximum level of crude proteins (19.99±1.4%) was found in the whole-body biochemical composition (% of wet weight) of Asian seabass juveniles in T3 treatment group reared at 22 ppt salinity (Table 4). Concerning Ash content, there was insigni cant among T5, T4, T3 and T2 in Ash content, but Ash content of T1 was 7.82±0.1 and signi cantly higher than the other treatments.

Discussion
As Asian sea bass L. calcarifer has great commercial values among the other marine sh species because of its usage as popular cultured species in most Southeast Asian countries including Thailand, Taiwan, Indonesia, Malaysia, Singapore and Hong Kong near coastal water, brackish and also in their freshwaters resources. Most countries have now used this cultured species for both development and research purposes in pond and cage culture. In this study, the means of physicochemical parameters were recorded in Table 3, did not show and marked variation and fall between the acceptable range for Asian sea bass growth and heath According to (Priestly et al. 2006; Hassan et al. 2020a; Kungvankij et al. 1986), who also found water pH ranged from 7.5 to 8.3, temperature 26 to 32°C, dissolved oxygen (DO) from 4.0 to 8.0 mg/L, Dissolved ammonia less than 0.02mg/L and salinity was considered as 10.0 to 31.0 ppt during culturing of fry and ngerling stages of L. calcarifer reared in concrete tanks near coastal waters in Thailand. Moreover, the nitrite and nitrate concentrations were comparatively low (> 5 mg/L) in ve experimental tanks of this study, because of addition of surface runoff water from rainfall bring pollutants includes domestic sewage wastes, agriculture and industrial wastes in sh ponds along with water resources used in sh culture and ammonium wastes from sh excreta released in sh ponds during culture of this species as in accordance with Christensen et al. (2012). Nitrate in ponds was higher than in canals because the decomposition of ammonia from sh waste to nitrate. However, nitrate was found in low concentration which meet the surface water quality criteria. Our results of water quality parameters was also found to be optimum for the growth of this species as reported by Jerry (2014). In addition, Abdullah et al. (2018) studied had shown the signi cant impact of dry and monsoon seasons on the water parameters includes salinity, dissolved oxygen concentration and turbidity of L. calcarifer culturing near Sri Tujuh lagoon of north east coast of Peninsular Malaysia.
In this study, the optimum salinity considered best for normal culture of seabass was 22 ppt (T3), which was very close to the results obtained by Ercan et al. (2015). Cheong (1989) had reported that as L. calcarifer is a euryhaline species; therefore, it can be easily cultured in both freshwater and brackish water, or even in seawater also. Therefore, this species had been successfully cultured in the freshwater ponds various regions of the world like in Thailand, and also in coastal water ponds like in Tahiti with salinity ranging from 10 to 35 ppt. Hence, most farmers commonly cultured L. calcarifer in ponds located along the sea coast with salinities ranging between 10 to 30 ppt, which is considered the optimum range for this species. Euryhaline sh undergo a crucial stage during their acclimatization in a hyperosmotic environment. There is a rapid increase in the gill-ion uid, followed by a rise in serum electrolytes and osmolality. In the present study, the acclimatization process for the juvenile sh was done to reduce the stress level of the sh during exposure to the different salinity treatments. The juvenile sh were not directly introduced to the various salinity treatments. Indeed, the salinity was decreased or increased slowly to the targeted level using a dropper to avoid sh startling. Thus, the juvenile can survive in a wide range of salinity without affecting its mortality rate (Cotton et al. 2003). In the present study, less mortality of juveniles was found for all the treatment groups (0 to 42 ppt salinity), which indicates that Asian seabass juveniles can resist abrupt hyper/hypoosmotic exposure. When sh have been questioned by medium salinity uctuations, the ion concentration in body uids has altered. This is in connection with the higher exchange layer on the skin and gut that lead to higher absorption of water (Shui et al. 2018).
Dendrinos & Thorpe (1985) had reported the signi cant impact of various salinity levels ranged from 0.5 to 33‰ on growth and body composition of European bass Dicentrarchus labrax and found that juveniles can survive in salinities between 5 to 33‰. They also observed its maximum growth rate at 30‰, which was decreased with decreasing salinity from 20 to 25‰, and even died in freshwater with 0‰ within few days. However, such changes in salinity levels have no impact on proximate composition of sh body muscles. In the present study, various salinity levels effected on the growth performance and survival rate, where the best growth performance and survival rate were recorded in T3 (22 ppt salinity). The previous study of Bernardino et al. (2016) had also found that salinity is the main factor affecting fecundity, osmoregulation, growth rate, feed consumption and survival of sh. Rearing sh in high salinity conditions has the potential to suppress the appetite of the sh. Li et al. (2008), Partridge et al. (2008) studies suggested that decreasing growth in increased salinity was related to the decrease in food consumption. The hyper-saline environment at 42 ppt salinity negatively effects on the growth rate, oxygen consumption, histological changes of hepatopancreas and survival as previously reported by Li et al. (2007). L. calcarifer at the extreme salinity range often increase their ability for osmoregulation; therefore, Partridge et al. (2008) observed that L. calcarifer if reared in salinity levels ranged between 0 to 36 ppt, then its expected speci c growth rate (SGR) of its juvenile stages can be obtained during its culture; however, if reared at 37 to 42 ppt salinities, than its may negatively affect its both growth performance and survival rates during culture.

Conclusion
From the above study, it was concluded that the highest growth and survival of Asian seabass L. calcarifer were observed at 22 ppt salinity as compared to the other salinity treatments. Therefore, we recommended 20 to 36 ppt salinity for commercial farming of this species under a closed aquaculture system. Sympathetic the physiological capacity of Asian Sea bass to adapt environmental salinity changes re ecting the sustainability of investment possibilities in the aquaculture eld. Using a distinct salinity concentration would maximize sea bass production and promote economic growth in aquaculture production.

Declarations Ethical approval and consent to participate
This study was carried out in strict accordance with the recommendations by the Local Ethical Committee for Experiments on Animals of the University of Karachi, Pakistan Funding There was no funding from any funding body or grant agency for conducting this study.

Availability of data and materials
All data analyzed during this study are included in this manuscript.

Consent to Publish
Not applicable. This manuscript did not contain data from any individual person.

Con ict of Interest
All authors declare no con icts of interest. All authors listed in the manuscript contributed and attest to the validity and legitimacy of the data and its interpretation and agree to its submission to your journal. 3 Nitrogen-free extract = 100 -(% protein + % lipid + % ber + % ash).