Evaluating the growth of genetically improved tilapia <i>Oreochromis niloticus</i> reared at different temperatures

<jats:title>Abstract</jats:title>                <jats:p>This study aimed to evaluate the growth and performance of genetically improved tilapia reared at different temperatures. Four hundred and eighty fingerlings of Genomar Supreme Tilapia, GST (8.39 ± 0.60 g) were equally separated into three indoor water recirculation systems maintained at 22, 26 and 30 ºC. Each of four tanks contained 500 liters with 40 fish per tank in natural photoperiod. The fish were fed ad libitum with the same feed for each growth phase, weighing the total feed supplied. After 30 min of feeding, leftover feed was collected, dried in an oven and weighed. Ten fish from each tank were weighed at days 1, 30, 60, 90, 120, 150, 180 and 210. Survival, weight gain, feed conversion and feed intake in each period were determined. Fish reared at 30 ºC and 26 ºC showed a higher specific growth rate than that of fish reared at 22ºC (P≤0.05). Feed intake increased along temperature and feed conversion and was poorest in fish reared at 26ºC (P≤0.05). Final fish weight estimates at day 210 by the Gompertz model were 597.84, 819.26 and 1079.39 g for 22, 26 and 30 ºC, respectively. At 30 ºC, fish had a higher absolute growth rate (7.76 g day<jats:sup>−1</jats:sup>) and lower weight (459.30 g) and age (95.85 days) at the inflection point. Tilapia at 22 ºC had a higher weight (539.57 g) and age (197 days) and lower absolute growth rate (4.52 g day<jats:sup>−1</jats:sup>). It was concluded that GST tilapia can potentially improve aquaculture in all Brazilian regions based on different rearing temperatures.</jats:p>

section_content: Brazilian aquaculture has rapidly increased over the past two decades through sustainably intensifying traditional farming systems, both pond and cage cultures. Through more experienced and efficient farming practices, productivity gain in these systems has been the main driver of the country's rapid growth in aquaculture production (Barroso et al., 2019). 

Freshwater fishes dominate finfish aquaculture in Brazil, and tilapias are the largest species group, accounting for nearly half of Brazil's aquaculture production (Barroso et al., 2019; Valenti et al., 2021). Brazil produced 486,155 tons of tilapia in 2020 with annual growth of 12. 5%, thus consolidating itself as the fourth largest producer in the world (Peixe Br, 2021). This increase is associated with such desirable characteristics as tolerance to different environments and production systems, resulting in its introduction in many subtropical and temperate regions of the world (Santos et al., 2013). In addition, the acceptability of fish as a meat source, combined with the search for healthy foods, has sparked the interest of producers for tilapia (Oreochromis niloticus) strains with superior performance (Santos et al., 2008). 

GIFT (genetically improved farmed tilapia) was the most popular selective breeding program carried out in Malaysia (Bentsen et al., 1998). Since the late 1990s, a variety of GIFT strains have been introduced in Brazil by private hatcheries or research institutes, including GIFT, GIFT Supreme, GIFT Spring and GIFT Aquamérica (Barroso et al., 2019). 

The Genomar Supreme Tilapia TM (GST) strain is the product of continuous GIFT breeding carried out in the Philippines (Santos et al., 2019). GST has shown higher performance than other strains in Brazil in our previous studies; however, few studies have reported on its performance in both warm and cold climates (Santos et al., 2013 (Santos et al.,, 2019)). The ability to genetically develop strains "preadapted" to local conditions is a major goal of fish breeding companies. 

In Brazil, fish farming can be found in cold temperatures below 22°C in the south and southeastern regions ------------*Work financed from FUNDAG (Fundação de Apoio à Pesquisa Agrícola) and São Paulo Research Foundation FAPESP, SP for Scientific Initiation Program, Process 2017/00880-1, 2017/01420-4 and 2016/13332-0. and temperatures higher than 28°C in the north and northeast. Water temperature is considered as the most important environmental factor because fish is a poikilothermic animal and its entire physiology depends on the changes in environmental temperature (Jobling, 1994). Therefore, fish has to spend extra energy for physiological homeostasis, maintaining the antioxidant and immune system (Buchtíková et al., 2011; He et al., 2015; Ma et al., 2015), which further have an effect on growth and survival (Baras et al., 2001; Qiang et al., 2012). Additionally, cold stress increases serum cortisol levels, inhibits lysozyme activity, decreases thyroxine levels and increases free radicals, causing oxidative damage in membranes and increased lipid peroxides (He et al., 2013 (He et al.,, 2015)). 

Generally, tilapia stop feeding when water temperature drops below 18°C, and they begin to die at temperatures below 14°C or 12°C (Tave at al., 1990; Sifa et al., 2002). According to Sifa et al. (2002), the GIFT strain is less tolerant to low temperature than the other two African strains, and the performance of genetically enhanced tilapia in cold climates, when developed in warmer regions, may be impaired. According to Qiang et al. (2018), a better understanding of regulation and response mechanisms of improved tilapia subjected to cold stress could have implications not only for reducing mortality during winter, but also improving the efficiency of fish farming. Winter temperature is low in the main tilapiarearing areas around the world, such as some regions in China or Brazil, and in recent years, more cold waves have occurred with temperatures that have substantially affected the survival and production of tilapia. 

According to Barroso et al. (2019), Brazil lacks diversified strains adapted to different farming conditions. While genetic improvement programs have been implemented for tilapia in Brazil, efforts have not resulted in commercial tilapia strains (Barroso et al., 2019). Thus, it would be desirable to correlate the growth of new strains of tilapia to different temperature gradients in the context of feed management plans to determine the maximum value from farmers' fish stocks. Accordingly, this study aimed to evaluate the growth and performance of genetically improved tilapia reared at different temperatures.

section_name: material and methods

section_content: This study was carried out at the Agency of Agribusiness Technology (APTA) in Presidente Prudente City (22°11' S and 51º23' W at an altitude of 458. 3 m), São Paulo State, Brazil. 

One thousand GST strain fingerlings from generation 25 (25 years from selection) weighing 0. 5 g were imported from the Philippines, and they were grown to a weight of 8 g in an indoor recirculating aquaculture system. In the importation process (Brazilian Ministry of Agriculture, Livestock and Supply PE. 1048/20216 and IBAMA 17BR022845/DF), these 1,000 live tilapia fingerlings were equally distributed into 4 polystyrene boxes (58 cm length × 41 cm width × 46 cm height) with each box containing 2 plastic bags of pure O 2 saturation. 

After this period, 480 fingerlings were equally separated into three indoor water recirculation systems maintained at 22, 26 and 30°C in four 500 L tanks, each containing 40 fish in natural photoperiod. Water recirculation was maintained by a pump enabling a constant flow of 1,500 L h -1 in each tank. The systems were supplied with temperature control (COEL z31) with heater or cooling equipment (Gelaqua 1 hp coolers), solids system decantation and pressurized filter (Atmam EF-6000uv). 

Water temperature of the recirculation systems was maintained at 22, 26 and 30°C based on different temperatures found in tilapia aquaculture systems in Brazil. Dissolved oxygen and temperature were monitored daily (Microprocessor Dissolved Oxygen Meter, Hanna 9146), and pH, ammonia, nitrite and nitrate were monitored weekly (Photometer System, Lovibond MD600). Dissolved oxygen was higher than 5. 0 mg L -1 during the entire study at all temperatures. Water quality analyses were performed weekly. 

Fish were fed three times a day ad libitum starting with commercial diet of 46% crude protein (CP) feed and 1. 7 mm pellets until fish weighed about 15 g, 42% CP with 2. 5 mm pellets until reaching 100 g, 36% CP with 3. 5 mm pellets until reaching 400 g and 32% CP with 4. 5 mm pellets until the end of the experiment. Feed intake (FI) was calculated as Fl = Tf -Lf, where Tf is the total feed supplied (g), and Lf is the leftover, dried and weighed (g). The interval between meals was 3 h and 30 min. After each meal, leftover feed was collected, dried in an oven (60°C) for 24 h, and weighed every day. 

Performance measurements, such as survival, weight gain and feed conversion, were determined at 1, 30, 60, 90, 120, 150, 180 and 210 days. Weight gain (WG) was calculated as WG = Wf -Wi, where Wf and Wi are final and initial weights (g), respectively. Feed conversion (FC) was calculated as

where Bf and Bi are final and initial biomass, respectively. Biomass (B) was calculated as B = W × N, where W represents mean fish weight, and N represents total number of fish in the tank. Specific growth rate (SGR) was calculated as SGR = 100(lnWf -lnWi)d -1, where Wf and Wi are the final and initial weight, and "d" is days of cultivation.
section_name: Location, fish and facilities

section_content: ANOVA was performed considering three treatments (temperature) and four replicates (tanks) at days 30, 60, 90, 120, 150, 180 and 210 of cultivation. In this case, 10 fish from each tank were weighed. Analyses of normality and homogeneity of variance were also performed. When temperature effect was significant, the analysis was supplemented with Tukey's multiple comparisons tests, using an alpha of 5%.
section_name: statistical analysis

section_content: Individual fish weight data from days 1, 60, 120, 150, 180 and 210 of cultivation (240 for each temperature) were fitted to the Gompertz model, given by y = Ae -Be-Kx, calculating the absolute (AGR) and relative (RGR) growth rates as AGR = Kyln(u -1 ) and RGR = Kln(u -1) and weight (WI) and age (AI) at the inflection point as WI = Ag -1 and AI = (lnB)K-1, respectively, according to Santos et al. (2013). 

The curve parameters for each temperature were compared using 95% confidence intervals. Equations and R² statistic were provided. The estimates were obtained by weighted least squares (Draper and Smith, 1998; Santos et al., 2008), using the Model procedure in SAS software (SAS ® OnDemand for Academics, SAS Institute Inc. ). 

Additionally, the mean (four tanks) amount of feed intake every day for each temperature was fitted to the Gompertz model, up to day 210, providing a feed table for GST tilapia rearing at 22, 26 and 30°C.
section_name: FC = total diet fed Bf -Bi

section_content: Mean pH and standard deviations (SD) were 6. 46 (0. 93), 6. 52 (0. 87) and 6. 35 (0. 74) at 22, 26 and 30°C, respectively. Maximum levels of ammonia were 0. 28 (0. 11), 0. 50 (0. 58) and 0. 25 (0. 36) mg L -1. Maximum nitrite concentrations were 0. 50 (0. 26), 0. 35 (0. 28) and 0. 18 (0. 09) mg L -1. Maximum nitrate concentrations were 56. 2 (35. 6), 24. 0 (13. 8) and 31. 3 (13. 6) mg L -1. 

The initial weights and standard deviation (SD) of the fingerlings at 22, 26 and 30°C were 8. 57 (0. 43), 7. 87 (0. 55), and 8. 75 (0. 50), respectively, and they were statistically similar (P>0. 05). Mortality was very low (1-2%) and similar at all temperature treatments (22, 26 and 30°C). 

Table 1 summarizes the performance of tilapia every 30 days of rearing at different temperatures. After day 30, the fish reared at 30°C had higher weight gain and better feed conversion compared to fish reared at 26 or 22°C (P≤0. 05). At day 120, the difference in weight gain between fish reared at 26°C relative to those reared at 22ºC was smaller (108. 27 g) than that between fish reared at 30 and 26°C (204. 36 g). After this period, the weight gains were similar among the treatments. Final weights showed a difference of about 33. 6% between fish reared at 26 and 30°C (856. 59 and 1144. 53 g) and a difference of 44. 9% between fish reared at 22 and 26°C (591. 05 and 856. 59 g). Feed conversion at day 30 and 60 was better in fish cultivated at 30°C, although at day 60, it was statistically similar to that at 22°C. Afterwards (day 90 until 210), fish cultivated at 22°C had better feed conversion, and this was associated with the specific growth phase. At day 60 of cultivation, the differences in fish size between groups are shown in Figure 1. Table 2 shows the overall performance at day 210 of tilapia cultured at the different temperatures. Gain weight increases with the increase of temperature of cultivation (P≤0. 05). Fish reared at 30 or 26°C showed higher specific growth rate (2. 29 and 2. 22 g day -1, respectively) than fish reared at 22°C (2. 02 g day -1 ). Feed intake increased along with temperature (P≤0. 05). Interestingly, feed conversion was better in tilapia reared at 22°C, followed by fish cultivated at 30°C (P≤0. 05). The highest feed conversion occurred in tilapia cultivated at 26ºC. 

Table 3 presents the estimated parameters of the Gompertz model. The fitting of weight data for tilapia cultured at 22, 26 or 30°C was good (high R² adjusted), showing applicable estimates and reliability. The estimate of asymptotic weight "A" was similar among the three temperatures. The growth rate "K" relative to maturity exhibited differences, and it was higher at 30°C (0. 0169) compared to that at 26°C (0. 0114) or 22°C (0. 0084). The final estimate at day 210 was 31. 7% higher for fish cultivated at 30°C compared to that of fish cultivated at 26°C and 37% higher in fish cultivated at 26°C compared to that of fish cultured at 22°C. The model fits are presented in Figure 2. 

Figures 3 and 4 present the AGR (g day -1 ) and RGR (%) of tilapia reared at different temperatures, respectively. Fish reared at 30°C showed the highest AGR. At this temperature, tilapia over 400 g had an AGR of about 8 g day -1, while at 26 and 22°C, it was 5. 5 and 4. 5 g day -1, respectively. At 30°C, fish also exhibited a higher RGR when compared to fish reared at 26 or 22°C. Weight, age at inflection point, and maximum AGR are presented in Table 4. At 30°C, fish had lower weight (459. 30 g) and age (95. 85 days), but higher AGR (7. 76 g day -1 ). Conversely, at 22°C, tilapia showed higher weight (539. 57 g) and age (197 days), but lower AGR (4. 52 g day -1 ). 

Figure 5 shows the feed intake of GST reared at different temperatures up to day 210, starting with fish weighing about 8. 0 g. At 22°C, tilapia started to ingest 4. 54% of biomass, and at day 210, intake was 1. 20%. At 26°C, the intake varied from 6. 20% at the beginning to 2. 10% at the end. At 30°C, the intake was 7. 51% at day 1 and 1. 44% at day 210. 22 ºC: y = 1,466. 7e (-5. 20e -0. 0084x ) R²Adj. = 0. 96 26 ºC: y = 1,312. 9e (-5. 19e -0. 0114x ) R²Adj. = 0. 98 30 ºC: y = 1,248. 5e (-5. 05e -0. 0169x ) R²Adj. = 0. 98 For many years, earthen ponds have been used to culture tilapia under extensive and semi-intensive systems; however, farmers and commercial entrepreneurs have, in many cases, shifted to more intensive systems in ponds, tanks and cages owing to the shortage of freshwater and widespread distribution of tilapia culture (El-Sayed, 2006). El-Sayed (2006) reviewed the effect of temperature on tilapia growth, considering optimum growth at 25-30°C. However, he reported that a tremendous difference in the growth and feed efficiency of tilapia might occur, even within this narrow range of water temperature. A wide range of culture systems and environments can be found between any two countries and within the same country, e. g., China and Brazil, or regions, such as sub-Saharan Africa. 

The performance of GST at 22°C in this study was similar to that of other Brazilian tilapia strains reared at 26°C, according to Santos et al. (2019). These results indicate that the breeding program conducted in the Philippines, as suggested earlier in this work, produced tilapia with better growth in cold temperatures. Thus, as reported by Ponzoni et al. (2008), it is important to define the appropriate environment for selection in any fish breeding program. 

On the other hand, even though Qiang et al. ( 2017 ) reported increased fish growth rates along with increased water temperature gradient, problems associated with high temperatures have emerged. These include increased susceptibility of tilapia to infectious diseases and liver damage and reduced lysozyme activity, immunoglobulin level and phagocytic function of white blood cells (Dominguez et al., 2005; Ndong et al., 2007; Zhu et al., 2016), as well as alteration in fatty acid composition and utilization (Samples et al., 1999; Ren et al., 2013). 

In our study, fish reared at 26°C showed performance closer to that of fish reared at 22°C than that of fish reared at 30°C. This could indicate that the breeding program in the Philippines, an equatorial country, was carried out in warmer waters, representing the optimum temperature for GST growth. Compared with our previous study in the same culture system, the results of this study show that GST performance at 22°C was similar to that of other strains cultured in Brazil at 28°C (Santos et al., 2019). 

Since Brazil is a continental country with many options for fish production systems, it is important to evaluate Genotype × Environment to gain insight on better performance in tilapia production since phenotype is a consequence of genotype under the influence of environment (Ponzoni et al., 2008; Oliveira et al., 2017). Similar studies were conducted in Malaysia (Khaw et al., 2009), the Philippines (Eknath et al., 2007) and Brazil (Oliveira et al., 2017) where the responses from GIFT fish were evaluated under different environmental conditions. 

The data presented in this study are important to tilapia farmers looking for maximum performance in a specific strain that can be reared under different conditions. If we consider a country such as Brazil, different feed tables suitable for different temperatures could have an additional application. It is always necessary to consider feed quality and the nutritional plan. In this case, the rearing system must also be given additional attention. In a recirculating aquaculture system, conditions are not always optimal, such as those involving constant handling (sampling and cleaning) and concentrations of ammonium, nitrite and nitrate, all of which can influence feed intake and, hence, fish growth. 

Here, the GST strain showed good performance and growth at 22°C. These results could be compared with data presented by Santos et al. (2019) who reported similar results with Brazilian strains cultured between 26 and 27°C. In recent years, genetics companies that work with tilapia, such as EW Group's GenoMar and Spring Genetics, have entered the Brazilian market, and each year, new generations of improved tilapia are made available to farms. Therefore, GST strain generation 25 seems promising for improved growth rate, weight gain and feed conversion in Brazilian tilapia cultures. 

While the focus has been primarily on increasing weight gain and fillet yield, genetic improvement activities in Brazil should concentrate efforts on such factors as cold resistance, disease resistance, and suitability for specific farming systems or arrangements (Barroso et al., 2019). Thus, more studies are necessary to evaluate different generations of the GST strain, considering these factors and the growth and performance of such generations in different culture systems, such as cages and ponds, under farming conditions.
section_name: results

section_name: Acknowledgements

section_content: Financial support was provided by the São Paulo Research Foundation -FAPESP, SP. Process 2017/00880-1, 2017/01420-4 and 2016/13332-0.
section_name: Acknowledgements

section_name: Ethical approval

section_content: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed by the authors.
section_name: Ethical approval