Aquaculture Research, 2003, 34, 241251

Low-temperature tolerance of Nile tilapia, Oreochromis niloticus: effects of environmental and dietary factors
H L Atwood1, J R Tomasso1, K Webb2 & D M Gatlin, III2
1 2

Department of Aquaculture, Fisheries & Wildlife, Clemson University, Clemson, SC, USA Department of Wildlife and Fisheries Sciences, Texas A & M University, College Station, TX, USA

Correspondence: Heidi L. Atwood, Department of Aquaculture, Fisheries and Wildlife, Clemson University, Clemson, South Carolina 29634, USA. E-mail: hatwood@clemson.edu

Abstract This study was conducted (1) to evaluate the effects of photoperiod (fixed vs. decreasing light), fish size (136 vs. 220 mm), dissolved ions (hardness and salinity) and diet (menhaden oil vs. coconut oilbased) on the tolerance (survival) of Nile tilapia, Oreochromis niloticus, to low temperatures (decreased by approximately 0.5 8C per day) and (2) to evaluate the effect of dietary fatty acid composition on selected physiological characteristics of Nile tilapia exposed to decreasing temperatures. Size significantly affected mortality, with smaller fish being less tolerant to low temperatures than larger fish. Results were equivocal in the photoperiod, dissolved ion and dietary lipid experiments, and were dependent on the method of data analysis employed. Diet significantly affected plasma osmolarity, with higher values in fish fed the menhaden-based diet. Haematocrit, serum glucose, sodium and cortisol concentrations, serum and splenic lysozyme activities, lymphocyte count and hepatosomatic index were not affected by diet. Haematocrit, white cell count and serum glucose and sodium concentrations were significantly affected by temperature, but serum and splenic lysozyme content, hepatosomatic index, and serum cortisol concentrations were not. The results of this series of experiments indicate that altering the environment or diet has little effect on the ability of Nile tilapia to survive low temperatures. Keywords: dietary lipid, dissolved ions, photoperiod, temperature, tilapia
2003 Blackwell Publishing Ltd

Introduction Tilapia (Oreochromis sp.) is a group of fish of growing importance in the aquaculture industry. In 1999, worldwide harvest of farmed tilapia surpassed 800 000 metric tons, and tilapia were second only to carp as the most widely cultured freshwater fish (Popma & Masser 1999). Nile tilapia (O. niloticus) constitute 90% of all tilapia cultured outside Africa (Popma & Masser 1999). Adaptation to relatively warm, stable ambient temperatures has limited production of tilapia to places where winter temperatures are above lethal minima or to part-year outdoor pond culture. Approximately 70% of yearround culture in the United States involves some portion of the winter months being spent indoors to prevent losses from excessive cold (ATA 1997). Resistance of tilapia to low temperature is genetically based (Cnaani, Gall & Hulata 2000), and tolerance is affected by abiotic factors such as season, water quality (hardness, salinity, dissolved oxygen, etc.) and biotic factors such as age, size, sex and condition of the fish (Chervinski 1982). Increasing dietary polyunsaturated fatty acid (PUFA) content has been shown to alter the fatty acid content of fish (Bell, Henderson & Sargent 1986; Henderson & Tocher 1987; Viola, Mokady, Behar & Cogan 1988; Seo, Chui & Hodson 1994; Kelly & Kohler 1999) and increase tolerance of some species to low temperatures (Farkas, Csengeri, Majoros & Olah 1980; Craig, Neill & Gatlin 1995). If environmental or dietary factors could be manipulated to increase tolerance of tilapia to low temperature, the
241

Low-temperature tolerance of Nile tilapia H L Atwood et al.

Aquaculture Research, 2003, 34, 241251

time spent in outdoor culture systems in temperate climates could be extended. Therefore, the objectives of this study were to evaluate the effects of specific environmental and dietary factors on tolerance of Nile tilapia to decreasing temperatures (approximately 0.5 8C per day) and to evaluate the response of selected physiological characteristics to decreasing temperature.

all experiments except for the dissolved ion experiment were 0.2 + 0.0 g L1 and < 20 mg L1 as CaCO3 respectively. During experiments, fish were fed twice daily until apparent satiation. During mortality experiments dead fish were removed daily and total length (TL) recorded.

Design of tolerance experiments Materials and methods Fish maintenance and experimentation Nile tilapia were obtained from the Clemson University Aquaculture Facility and maintained in a 1500-L recirculating water system until used in experiments. During the holding period, and during all environmental factor experiments, a diet of trout feed (crude protein $38%, crude fat $ 8%, crude fibre # 4%; Zeigler Brothers, Gardners, PA, USA) was fed twice daily to apparent satiation. Water quality in the holding system was: temperature 24.2 + 1.4 8C (mean + SD); pH 6.6 + 0.5; salinity 0.4 + 0.2 g L1 (synthetic sea salt; Instant Ocean, Aquarium Systems, Mentor, OH, USA); total ammonia-N 0.7 + 1.0 mg L1; nitrite-N 0.1 + 0.2 mg L1; dissolved oxygen 6.5 + 0.8 mg L1. Photoperiod for the holding system was 12 h light to 12 h dark (12 L:12 D). All experiments were conducted in 475-L recirculating systems (Living Streams: Frigid Units, Toledo, OH, USA). Each recirculating system was equipped with a thermostatically controlled chiller and immersion heater. Photoperiod was set by electric timer, and water was continuously aerated using air stones. During experiments, temperature and salinity were monitored daily (model 30/10FT temperature/salinity meter; Yellow Springs Instrument Company, Yellow Springs, OH, USA). Temperature was validated against an ANSI/SAMA standard calibrated thermometer. The pH was monitored at least weekly (Accumet model 915 pH meter; Fisher Scientific, Pittsburgh, PA, USA). Ammonia and nitrite were monitored at least weekly according to APHA (1989). Dissolved oxygen was monitored weekly (model 58 dissolved oxygen meter; Yellow Springs Instrument Company). Water quality in the recirculating systems for all experiments was: pH 7.2 + 0.7; dissolved oxygen 8.9 + 1.6 mg L1; nitrite-N 0.8 + 1.4 mg L1; and ammonia-N 0.3 + 0.5 mg L1. Mean salinity and hardness for
242

To determine the effect of photoperiod on susceptibility to decreasing temperature, five recirculating systems in each of two rooms were each stocked with 10 fish (TL 111 + 8.1 mm) transferred from the holding system. The photoperiod was initially set to 12 L:12 D. Fish were allowed to acclimate to the experimental systems for 6 days at 23.9 + 0.4 8C. One room was kept on the 12 L:12 D schedule, and the temperature lowered. The photoperiod in the second room was altered to simulate a time/temperature compression (~ 30 days) similar to local conditions of decreasing light and temperatures observed in the autumn months (AugustNovember). The temperature regime was adopted to allow for temporal physiological acclimation while providing an estimate of mean lower-lethal temperature limits. This schedule resulted in a decrease of 0.5 8C day1 (r2 0.981). Temperatures were decreased until all fish died. To determine the effect of fish size on susceptibility to decreasing temperature, five recirculating systems were divided into two sections and each was stocked with five large fish (TL 220 + 31.1 mm) in one section and 10 small fish (136 + 11.7 mm) in the second section. Fish were allowed to acclimate to the experimental systems for 6 days at 24.8 + 0.9 8C. Water temperatures were then decreased by 0.5 8C day1 (r2 0.985) until all fish died. To determine the effects of salinity and hardness on susceptibility to decreasing temperature, 10 recirculating systems were each stocked with 10 fish (TL 114 + 8.4 mm). Each of two treatment groups was randomly assigned to three of the 10 systems and four systems were maintained as controls. The salinity treatment was 8.7 + 0.6 g L1 synthetic sea salt (Instant Ocean, Aquarium Systems, Mentor, OH, USA), chosen because it is close to isosmotic with fish plasma (Beamish 1970; Woo, Ng, Leung & Chow 1997). The hardness treatment was 94.4 + 9.7 mg L1 calcium (as calcium sulphate). Salinity and hardness were similar to

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Aquaculture Research, 2003, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al. Table 1 Composition (%) of the experimental diets fed to Oreochromis niloticus
Diet Ingredient Casein Gelatin Dextrin Corn oil Menhaden oil Coconut oil Vitamin premix* Mineral premix CMC Celufil Menhaden oil 28.5 7.0 23.5 3.0 7.0 0.0 3.0 4.0 2.0 22.0 Coconut oil 28.5 7.0 23.5 3.0 0.0 7.0 3.0 4.0 2.0 22.0

that of quarter-strength seawater. Control water was soft (total hardness 18.7 + 2.0 mg L1 as CaCO3; calcium 0.6 + 0.9 mg L1; salinity 0.1 g L1). Fish were allowed to acclimate to experimental conditions for 7 days (for 4 days in their treatments) at 25.1 + 0.4 8C. Water temperatures were then decreased by 0.5 8C day1 (r2 0.966) until all fish died. To determine the effect of diet on susceptibility to decreasing temperature, two different experimental diets were prepared. Menhaden and coconut oil diets were formulated and prepared at Texas A & M University. Diets were formulated to contain 34% crude protein from casein (80%) and gelatin (20%). Either coconut oil (7%) or menhaden oil (7%) was supplemented to develop diets containing distinctive fatty acid profiles. Coconut oil provided high levels of 12- and 14-carbon saturated fatty acids and menhaden oil provided high levels of highly unsaturated (n-3) fatty acids. Corn oil (3%) was added to both diets to meet essential fatty acid requirements (Takeuchi, Satoh & Watanabe 1983). Specifics of the diet composition are listed in Table 1. Vitamin and mineral premixes were added to the diet formulation to ensure that nutritional requirements were met, and dextrin was included to increase available energy to 3.2 kcal g1. Diets were frozen and shipped to Clemson University, where they were stored frozen until needed. The fatty acid composition of both diets is listed in Table 2. In this experiment, 10 recirculating systems were each stocked with 10 fish (TL 128 + 11.8 mm). Each of the two experimental diets was randomly assigned to five systems. Fish were allowed to acclimate to the experimental systems for 4 days and to their assigned diet for an additional 14 days at 23.9 + 0.4 8C. Temperature was then decreased 0.6 8C day1 (r2 0.983) until all fish died. Fish were collected when they died, total length was measured to the nearest millimetre and they were then frozen at 45 8C, sorted by date of death, system number and treatment and shipped on dry ice to Texas A & M University for fatty acid analysis.

*Vitamin premix contains (as g kg1) 50.0 ascorbic acid, 5.0 dicalcium pantothenate, 36.2 choline chloride, 5.0 inositol, 2.0 menadione sodium bisulphite, 5.0 niacin, 1.0 pyridoxine HCl, 3.0 riboflavin, 0.5 thiamine mononitrate, 8.0 dl-alpha-tocopherol acetate (250 IU g1), 0.2 vitamin A palmitate (500 000 IU g1), 5.0 biotin, 180.0 folic acid, 0.2 vitamin B12, 0.2 cholecalciferol (40 IU mg1), and 976.6 cellulose. Mineral premix contains (as g kg1) 136.0 calcium phosphate monbasic, 348.49 calcium lectate, 5.0 ferrous sulphate, 132.0 magnesium sulphate heptahydrate, 240.0 potassium phosphate dibasic, 88.0 sodium phosphate monobasic, 45.0 sodium chloride, 0.15 aluminum chloride, 0.15 potassium iodide, 0.50 cupric sulphate, 0.70 manganese sulfate, 1.0 cobalt chloride, 3.0 zinc sulphate heptahydrate, and 0.011 sodium selenite. Carboxymethyl cellulose.

Design of physiology experiment The second part of the diet experiment was conducted to determine the effect of diet on selected physiological characteristics of Nile tilapia subjected to decreasing temperatures. Six recirculating systems were each stocked with 10 fish

(167 + 14.7 mm). Each of the experimental diets was randomly assigned to three systems. Fish were allowed to acclimate to the experimental system for 2 days and to their assigned diet for an additional 14 days at 25.1 + 0.4 8C. Water temperatures were then decreased by 0.5 8C day1 (r2 0.941) until sampling was complete or all remaining fish were dead. Two fish from each system (six fish per treatment) were collected at approximately weekly intervals during the experiment (five sample days corresponding to 25, 20, 17, 14, 9 8C). On sampling days, fish were netted and anaesthetized individually in buffered MS222 (100 mg L1; tricaine methanesulphonate; Argent Chemical, WA, USA) prepared in water from their system. Blood was collected from the caudal sinus into a non-heparinized Vacutainer tube and allowed to clot. Serum was collected following centrifugation and stored at 45 8C for later determination of cortisol, glucose and sodium concentrations and
243

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al.

Aquaculture Research, 2003, 34, 241251

Table 2 Fatty acid composition (g kg1 fatty acid) of experimental diets and whole body of Oreochromis niloticus fed experimental diets*. Different superscripts (a, b) denote difference in composition (P # 0.05)
Diet Dietary fatty acids Coconut oil neutral Menhaden oil neutral Coconut oil polar Menhaden oil polar Whole body neutral lipid Coconut oil Menhaden oil Mean square Whole body polar lipid Coconut oil Menhaden oil Mean square Sat. Mono. PUFA (n-3){ (n-6)** (n-3)/(n-6)

6.93 2.89 4.98 2.82 4.09a 3.29b 0.84 3.52a 3.24b 0.36

1.29 2.56 2.08 2.53 3.26a 3.39a 0.24 2.15a 2.01a 0.26

1.79 4.55 2.93 4.65 2.66a 3.32b 0.50 4.32a 4.76b 0.54

0.03 1.99 0.31 2.20 0.88a 1.49b 0.33 1.89a 2.93b 0.67

1.75 2.08 2.62 2.00 1.32a 1.42b 0.05 1.32a 1.29a 0.06 0.67 1.05

1.43 2.27

*Where applicable values are means of analysis for each diet; means of two fish from each of four or five replicate groups. Total saturated fatty acids. Total monounsaturated fatty acids. Total polyunsaturated fatty acids. {Total (n-3) fatty acids. **Total (n-6) fatty acids. Normal ratio range for neutral lipid in freshwater fish is 1.083.3; normal ratio range for polar lipid in freshwater fish is 1.62.0 (Henderson & Tocher 1987).

lysozyme content. Heparinized microcapillary tubes were used to collect blood from the severed caudal peduncle for haematocrit, lymphocyte count and osmolality. Livers were removed and weighed to the nearest 0.001 g and spleens removed and stored at 45 8C for later analysis of lysozyme content. Fish were weighed to the nearest 0.01 g and total length determined to the nearest millimetre. Analyses To determine fatty acid profiles, individual whole fish were dipped in liquid nitrogen and pulverized. Frozen pieces were then ground to a homogeneous consistency and samples analysed for dry matter and ash content in accordance with AOAC (1990) methods. Total lipid (Folch, Lees & Stanley 1957) and fatty acid composition of the neutral and polar lipid fractions (Satoh, Poe & Wilson 1989) were determined. Haematocrit was determined by centrifugation of a heparinized microhaematocrit tube, and one drop of heparinized whole blood was smeared on a glass slide and stained with Diff-Quik (Baxter Diagnostics, Pittsburgh, PA, USA) for lymphocyte count. Osmolality was measured using heparinized fresh
244

plasma with a vapour pressure osmometer (model 5500, Wescor, Logan, UT, USA). Hepatosomatic index (HSI) was determined by dividing fresh liver weight by fish weight and then multiplying by 100. Cortisol was measured in serum by enzyme immunoassay (Diagnostic Systems Laboratories, Webster, TX, USA). Glucose was measured in serum by colorimetric enzymatic assay (Sigma Diagnostics, St. Louis, MO, USA). Sodium concentration in serum was measured by flame emission spectroscopy using an atomic absorption spectrometer (Perkin Elmer Aanalyst 800, Shelton, CT, USA). Serum and splenic lysozyme content in individual samples were measured by the turbidometric method according to Parry, Chanian & Shahani (1965). Lysozyme content was determined by activity and one unit of lysozyme activity was defined as the amount of sample causing a decrease in absorbance of 0.001 min1. Thermal tolerance data were analysed by logistic regression analysis, P # 0.05 (comparison of the slopes of lines) (SAS Version 8.0; SAS Institute, Cary, NC, USA), using per cent cumulative mortality for each treatment and either mortality temperature (temperature at death, TAD) or the cumulative temperature effect variable (cooling degree-day, CDD).

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Aquaculture Research, 2003, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al.

Cooling-degree day is an empirical estimation of cold tolerance. This variable represents the sum of the days the fish survived multiplied by the difference between daily temperature and the initial temperature for each fish (Cnaani et al. 2000). These two perspectives on temperature exposure were compared for similarity in detecting differences between treatment groups. Physiological data were analysed in a three-step manner. First, a two-way (temperature feed) analysis of variance (anova) with interaction analysis was conducted ( JMP Version 2; SAS Institute). If no interaction was indicated, the two-way anova was repeated without the interaction term. When a significant effect of temperature was observed, a oneway anova was performed followed by a Tukey Kramer pairwise comparison among treatment means. Significance was set at P # 0.05 for all analyses. Results and discussion Visually observed changes associated with decreasing temperature included changes in behaviour and coloration. All fish exhibited a general decline in activity, darkening of body coloration and an abrupt loss of equilibrium. As temperatures declined, fish stayed close to the bottom and oriented themselves facing into the current. All tilapia ceased feeding prior to disorientation or death, although the point at which they ceased to feed varied from 18 8C to 13 8C. First mortality in any experiment occurred at 10.6 8C, and all fish succumbed to decreased temperature by 6.8 8C. The results of assessment of cumulative mortality by TAD or CDD were mixed. During the photoperiod experiment the first fish died at 9.5 8C (12 L:12 D) and 9.8 8C (altered). The last fish died at 8.3 8C (12 L:12 D) and 8.0 8C (altered). The temperature at which 50% of the fish died (LT50) for each treatment was 9.0 8C (12 L:12 D) and 9.2 8C (altered). Fish in one recirculating system in the decreasing photoperiod room had to be euthanized during the experiment because of a fungal infection and were not included in the analysis. The effect of altered photoperiod was significant based on CDD (P 0.0001) but not on TAD (P 0.7364). Photoperiod and temperature have been shown to significantly affect fish physiology and behaviour. Photoperiod in conjunction with temperature has been evaluated in several contexts in a number of species, including growth of embryonic dogfish,

Scyliorhinus canicula (Thomason, Conn, Le Comte & Davenport 1996), growth of larval greenback flounder, Rhombosolea tapirina (Hart, Hutchinson & Purser 1996), reproduction in yellow perch, Perca flavescens (Ciereszko, Dabrowski, Ciereszko, Eberling & Ottobre 1997), swimming performance of white crappie, Pomoxis annularis (Smiley & Parsons 1997), seed production in tilapia, O. spilurus and O. niloticus (Ridha, Cruz, Al-Ameeri & Alahmed 1998; Ridha & Cruz 2000), and growth of Atlantic halibut, Hippoglossus hippoglossus ( Jonassen, Imsland, Kadowaki & Stefansson 2000). During the experiment to determine if fish size affected low-temperature tolerance, the first fish died at 10.6 8C (small) and 9.6 8C (large) and the last died at 8.2 8C (small and large). The LT50 was 9.0 8C for small fish and 9.1 8C for large fish. Fish in one recirculating system had to be euthanized during the experiment because of a fungal infection, which was first observed in the smaller fish, and these fish were not included in the analysis. Both methods of analysis found size to make a difference. Small fish were more susceptible to decreasing temperatures than larger fish, but the CDD approach indicated effects to be more significant (P 0.0012) than did the TAD approach (P 0.0359). Other studies with Nile tilapia have seen similar susceptibility in smaller fish. A sudden drop in temperature in culture ponds caused small Nile tilapia (5 cm) to lose equilibrium and roll over before larger fish (17 cm) (Yashouv 1960). The survival rate of large Nile tilapia fry was significantly higher than that of smaller fry under winter culture conditions in northern Vietnam (Dan & Little 2000). Behrends, Kingsley & Bulls (1990), however, found a lack of correlation between fish size and tolerance in tilapia of several species. During the dissolved ions experiment, the first fish died at 7.9 8C (hard water), 8.6 8C (soft water) and 8.9 8C (saline water). The last fish died at 7.1 8C (hard water), 6.8 8C (soft water) and 6.9 8C (saline water). The LT50 for each treatment was 8.1 8C (hard water), 8.1 8C (soft water), and 8.0 8C (saline water). The effect of salinity on mortality was found to be significant when assessed by CDD (P 0.0001), salinity having less ameliorative effect than hardness (P 0.0731) vs. soft water. Neither salinity (P 0.3977) nor hardness (P 0.3977) was different from soft water when assessed by TAD. Salinity and hardness have been found to be effective in improving tolerance to cold in some species (Craigie 1963; Allanson, Bok & van Wyk
245

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al.

Aquaculture Research, 2003, 34, 241251

1971; Chervinski & Lahav 1976; Stauffer, Vann & Hocutt 1984; Jennings 1991) but not in others (Stauffer 1986; Zale & Gregory 1989). It has been observed that osmoregulatory failure contributes to temperature-induced mortality during environmentally realistic decreases in temperature (Umminger 1969; Allanson et al. 1971). By addition of salts to the water it was hoped that osmotic stress would be reduced, but the fish so treated actually succumbed at a higher or equal temperature (depending on the analysis used) than those in the other two treatments. In the diet experiment, the first fish died at 12.3 8C (menhaden oil) and 10.9 8C (coconut oil) and the last fish at 7.0 8C (menhaden oil) and 7.5 8C (coconut oil). The LT50 for each treatment was 7.6 8C (menhaden oil) and 8.1 8C (coconut oil). One coconut feed replicate was removed from the experiment because of chilling unit failure and was not included in the analysis. Results differed by the method of analysis used. The effect of feed type was significant based on TAD (P 0.0003) but not by CDD (P 0.2151). Given the better predictive value of the CDD method and the small (0.5 8C) difference in LT50 values, it is likely that the diets had little effect on the tolerance of tilapia to low temperature. Previous studies have shown a change in muscle and liver fatty acid composition as a result of diet in hybrid tilapia (O. niloticus O. aureus; Farkas, Csengeri, Majoros & Olah 1980; Huang, Huang & Hou 1998; Chou, Shiau & Hung 2001) and in several other species (Satoh et al. 1989; Craig et al. 1995). However, only some incorporation of polyunsaturated fatty acids (PUFAs) was seen in hybrid tilapia after feeding a prepared diet (7% fish oil), but no change in total body fat or fatty acid composition was observed during a 21-day cold temperature exposure (Viola et al. 1988). In this experiment, fatty acid composition of Nile tilapia changed in less than a month and menhaden oil-fed fish incorporated a higher percentage of PUFA vs. saturated fatty acids than did fish fed the coconut oil diet (Table 2). Fish fed the coconut oil diet had higher levels of (n-6) fatty acids, and fish fed the menhaden oil diet had higher levels of (n-3) fatty acids. The (n-3)/(n-6) ratio was higher for menhaden oil-fed fish than for those fed coconut oil. The (n-3) to (n-6) ratio for both neutral (menhaden 1.05; coconut 0.66) and polar lipids (menhaden 2.27; coconut 1.43) were comparable with the normal range for freshwater fish (Table 2; Henderson & Tocher 1987). Some fish fed diets
246

high in (n-3) fatty acids have demonstrated increased tolerance to low temperatures (Craig et al. 1995). When comparing the slope of the line for each treatment, the CDD approach appeared to be more sensitive to temperature effect than the TAD approach. The model based on cumulative effects of temperature should better reflect the effect on the animals and is probably a better method for determining subtle differences in resistance or tolerance to low temperatures. Exposure to low temperatures not only affects mortality directly but also appears to depress the immune system of these warmwater fish. The fungus Saprolegnia is common in tilapia held at low temperatures (Popma & Lovshin 1996; Behrends et al. 1990). Fish in one recirculating system in the first experiment and one in the third experiment were euthanized after a fungal infection became apparent. Plasma osmolarity (Fig. 1) was significantly affected by diet (P 0.0401) but not by temperature (P 0.0605). In acute studies (approximately 1 8C h1 decrease), with Nile tilapia, plasma osmolarity was observed to decrease in response to decreasing temperatures (Sun, Chen & Chang 1992), or in hybrid tilapia, to not change at all (Al Amoudi, El-Sayed & El-Ghobashy 1996). Mozambique tilapia, Oreochromis mossambicus, subjected to decreasing temperatures at a rate of 1 8C 6 h1 showed no change in osmolarity until 11 8C, when all fish were impaired (Allanson et al. 1971) A change in osmolarity may indicate incipient osmoregulatory failure and loss of serum electrolytes to the environment. The water temperature in this experiment was decreased more slowly than in that of Sun
Menhaden Coconut 370 Osmolarity (mmol/kg) 360 350 340 330 320 310 300 25 20 15 Temperature ( C)
Figure 1 Plasma osmolarity (mean + SD) measured in Nile tilapia, Oreochromis niloticus, exposed to decreasing temperatures (0.5 8C day1).

10

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Aquaculture Research, 2003, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al.

et al. (1992) or Allanson et al. (1971), apparently allowing time for the fish to compensate and maintain osmolarity at near normal levels (338 + 11.8 mmol kg1 measured in Nile tilapia maintained at 25 8C in this laboratory). Haematocrit (Fig. 2) was not significantly affected by diet (P 0.7390), but was affected by temperature (P 0.0083). There also was an interaction between temperature and diet (P 0.0101). This is consistent with results seen in acute studies with channel catfish (Bly & Clem 1991), but is inconsistent with the response seen in acute studies with Nile tilapia (Sun et al. 1992), in which haematocrit remained relatively consistent. This lack of response may reflect the acute nature of the temperature change and short duration of the Sun et al. (1992) study. Decrease in haematocrit has been postulated to be the result of a decrease in erythropoiesis or shrinkage of red blood cells (as seen in rainbow trout, Oncorhynchus mykiss; Sovio, Nyholm & Westman 1973). A lower haematocrit may also be the result of decreased dependency on bound oxygen transport as a result of lower oxygen requirements at lower temperatures and increased solubility of oxygen in plasma. The opposite effect has been seen with increasing environmental temperature, with an increase in red blood cell production to meet increasing oxygen demand (Chudzik & Houston 1983). Lymphocyte count (Fig. 3) was not significantly affected by diet (P 0.3119) but was significantly affected by temperature (P 0.0018). This is consistent with decreases in white cell numbers and per cent white cells seen in Nile tilapia (Sun

et al. 1992) and in other species exposed to decreasing temperatures (Muona & Sovio 1992; Hrubec, Robertson & Smith 1997). Serum cortisol concentrations were not significantly affected by either diet (P 0.6918) or temperature (P 0.4834). Mean values ranged from 6.5 + 5.4 mg dL1 (14 8C) to 20.0 + 4.5 mg dL1 (9 8C) for menhaden diet fish and from 3.2 + 2.6 mg dL1 (20 8C) to 20.4 + 17.8 mg dL1 (17 8C) for coconut diet-fed fish. Values were consistent with those observed in blue tilapia (Kindle & Whitmore 1986) but 10-fold higher than for those recorded for resting, unstressed Nile tilapia (Barcellos, Nicolaiewsky, de Souza & Lulhier 1999). In blue tilapia (Kindle & Whitmore 1986) and channel catfish, Ictalurus punctatus (Strange 1980), animals acclimated to lower temperatures had higher resting cortisol levels than animals at higher temperatures. Other species acclimated to different temperatures have shown no differences in plasma cortisol levels (chinook salmon, Oncorhynchus tshawytscha, Strange et al. 1977; goldfish, Carasius auratus, Peter et al. 1978; largemouth bass, Micropterus salmoides, Carmichael, Tomasso, Simco & Davis 1984). Serum glucose concentrations (Fig. 4) were not affected by diet (P 0.9336), but increased significantly with decreasing temperature (P 0.0001). This is consistent with results observed in channel catfish (Strange 1980), blue tilapia (Kindle & Whitmore 1986), Nile tilapia (Sun et al. 1994), and hybrid striped bass, Morone chrysops Morone saxatilis (Tomasso, Isely & Tomasso 1996). The
25 20 15 10 5 0 5 25 20 15 Temperature ( C)
Figure 3 Lymphocyte count 103 RBC (mean + SD) measured in Nile tilapia, Oreochromis niloticus, exposed to decreasing temperatures (0.5 8C d1). In the upper-left, sampling temperatures with statistically similar mean responses (pooled dietary data) share a common line (TukeyKramer test).

9, 14, 17, 20, 25 9, 14, 17, 20, 25 Menhaden Coconut 35 30 25 20 15 25 20 15 10 Temperature ( C) 5 Haematocrit (%)

Menhaden Coconut

10

Figure 2 Haematocrit (mean + SD) measured in Nile tilapia, Oreochromis niloticus, exposed to decreasing temperatures (0.5 8C day1). In the upper-left, sampling temperatures with statistically similar mean responses (pooled dietary data) share a common line (TukeyKramer test).

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

247

Lymphocytes

Low-temperature tolerance of Nile tilapia H L Atwood et al.

Aquaculture Research, 2003, 34, 241251

Glucose (mg/dl)

3.50 3.30 9, 14 9, 14, 17, 20, 25 14, 17, 20, 25 25 20 15 Temperature ( C) 10 5 3.10 3.90 2.70 2.50

25

20

15 Temperature ( C)

10

Figure 4 Serum glucose concentrations (mean + SD) measured in Nile tilapia, Oreochromis niloticus, exposed to decreasing temperatures (0.5 8C day1). Top left: sampling temperatures with statistically similar mean responses (pooled dietary data) share a common line (TukeyKramer test).

Figure 5 Serum sodium concentrations (mean + SD) measured in Nile tilapia, Oreochromis niloticus, exposed to decreasing temperatures (0.5 8C day1). Bottom left: sampling temperatures with statistically similar mean responses (pooled dietary data) share a common line (TukeyKramer test).

increase in serum glucose concentrations at low temperatures may represent a form of thermal acclimation by increasing substrate concentration to compensate for lower enzymatic reaction rates. Hyperglyaecemia also helps maintain serum osmolarity in freshwater fish during low-temperature exposure (Kindle & Whitmore 1986). Serum sodium concentrations (Fig. 5) were not affected by diet (P 0.0606), but decreased significantly with temperature (P 0.0009). This is consistent with observations of Mozambique tilapia (Allanson, Bok & van Wyk 1971) subjected to a temperature decrease of 1 8C/6 h. No other studies were found that determined sodium concentration, but similar findings were observed for chloride concentration at low temperature in Mozambique tilapia (Allanson et al. 1971). No change in sodium concentrations was observed during acute decreases in temperature in blue tilapia (Kindle & Whitmore 1986) or Nile tilapia (Sun et al. 1994). Hepatosomatic index (HSI) was not affected by diet (P 0.1376) or temperature (P 0.0655). Mean values ranged from 0.8 + 0.1 (20 8C) to 1.6 + 0.8 (17 8C) for menhaden diet fish and from 1.0 + 0.3 (20 8C) to 1.8 + 0.2 (14 8C) for coconut diet-fed fish. The HSI has been observed to decrease in other species subjected to low temperatures (Craig et al. 1995; Seddon & Prosser 1997). Decreases in HSI are often seen in stressed fish (Lee et al. 1983; Ram & Singh 1988), ostensibly because chronic stress imposes an energy demand on the fish that can be correlated to energy source depletion (i.e. liver glycogen).
248

Serum lysozyme and splenic lysozyme content were not affected by either diet (serum, P 0.0978; spleen, P 0.4111) or temperature (serum, P 0. 1799; spleen, P 0.0862). Considerable fish to fish variability was observed in both serum and splenic lysozyme content. Mean serum lysozyme values ranged from 150.0 + 70.7 U mL1 (25 8C) to 275.0 + 68.1 U mL1 (20 8C) for menhaden diet fish and from 75.0 + 35.4 U mL1 (14 8C) to 191.7 + 118.2 U mL1 (20 8C) for coconut diet fish. Mean splenic lysozyme values ranged from 5.3 103 + 2.3 U g1 (14 8C) to 36.0 103 + 21.2 U g1 (20 8C) for menhaden diet fish and from 3.3 103 + 3.1 U g1 (14 8C) to 22.0 103 + 15.9 U g1 (25 8C) for coconut diet fish. Lysozyme is present in almost all body fluids and leucocyte-rich tissues such as the spleen, kidney and gut. Monocytes, macrophages and neutrophils are thought to be the predominant origin of lysozyme (Ellis 1990). Serum lysozyme content was observed to be lower in Nile tilapia incubated at 15 8C rather than 30 8C (Chiayvareesajja, Red, Eknath, Danting, De Vera & Bentsen 1999) and in plaice, Pleuronectes platesas, maintained at 5 8C for 3 months (Fletcher & White 1976). Lysozyme activity in fish is modified by stress conditions and the variation is relevant to the intensity and duration of stress conditions (Mock & Peters 1990). Although it is appealing as a standard indicator of non-specific humoral immune response, it is influenced by body weight, sex, stage of sexual maturation (Chiayvareesajja et al. 1999) and even social status among groups of individuals

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Sodium (g/L)

9, 14 9, 14, 17 14, 17, 20, 25 16, 20, 25

Menhaden Coconut

360 320 280 240 200 160 120 80 40 0

Menhaden Coconut

4.10 3.90 3.70

Aquaculture Research, 2003, 34, 241251

Low-temperature tolerance of Nile tilapia H L Atwood et al. Federation (1989) Standard Methods for Examination of Water and Waste Water, 17th edn. American Public Health Association, Washington, DC. ATA (American Tilapia Association) (1997) 1996 tilapia situation and outlook report. Aquaculture Magazine (SeptemberOctober), 612. Barcellos L.J.G., Nicolaiewsky S., de Souza S.M.G. & Lulhier F. (1999) Plasmatic levels of cortisol in the response to acute stress in Nile tilapia, Oreochromis niloticus (L.), previously exposed to chronic stress. Aquaculture Research 30, 437444. Beamish F.W.H. (1970) Influence of temperature and salinity acclimation on temperature preferenda of the euryhaline fish Tilapia nilotica. Journal of the Fisheries Research Board of Canada 27, 12091214. Behrends L.L., Kingsley J.B. & Bulls M.J. (1990) Cold tolerance in maternal mouthbrooding tilapias: phenotypic variation among species and hybrids. Aquaculture 85, 271280. Bell M.V., Henderson R.J. & Sargent J.R. (1986) The role of polyunsaturated fatty acids in fish. Comparative Biochemistry and Physiology 83B, 711719. Bly J.E. & Clem L.W. (1991) Temperature-mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish. Veterinary Immunology and Immunopathology 28, 365377. Carmichael G.J., Tomasso J.R., Simco B.A. & Davis K.B. (1984) Confinement and water quality-induced stress in largemouth bass. Transactions of the American Fisheries Society 113, 767777. Caruso D. & Lazard J. (1999) Subordination stress in Nile tilapia and its effect on plasma lysozyme activity. Journal of Fish Biology 55, 451454. Chervinski J. (1982) Environmental physiology of tilapias. In: The Biology and Culture of Tilapias (ed. by R.S.V. Pullin & R.H. Lowe-McConnell), pp. 119128. ICLARM (International Center for Living Aquatic and Marine Resources) Conference. Proceedings 7. International Center. For Living Aquatic Resources Management, Manila, Philippines. Chervinski J. & Lahav M. (1976) The effect of exposure to low temperature on fingerlings of local tilapia (Tilapia aurea) (Steindachner) and imported tilapia (Tilapia vulcani) (Trewavas) and Tilapia nilotica (Linne) in Israel. Bamidgeh 28, 2529. Chiayvareesajja J., Red K.H., Eknath A.E., Danting J.C., De Vera M.P. & Bentsen H.B. (1999) Genetic variation in lytic activities of blood serum from Nile tilapia and genetic associations with survival and body weight. Aquaculture 175, 4962. Chou B., Shiau S. & Hung S. (2001) Effect of dietary cod liver oil on growth and fatty acids of juvenile hybrid tilapia. North American Journal of Aquaculture 63, 277284. Chudzik J. & Houston A.H. (1983) Temperature and erythropoiesis in goldfish. Canadian Journal of Zoology 61, 13221325.

(Caruso & Lazard 1999). It was hoped that the inclusion of menhaden oil in the diet might have improved lysozyme content as macrophage activity was improved in channel catfish even at lower than physiologically optimal temperature (Sheldon & Blazer 1991). This study verifies that the fatty acid profile of Nile tilapia can be affected by diet, and demonstrates that the profile can change more quickly than previously reported. However, changes in fatty acid profiles that are associated with increased tolerance of low temperatures in red drum, Sciaenops occellatus (Craig et al. 1995), were not associated with increased tolerance to low temperatures in Nile tilapia as measured by survival and specific physiological responses. There is great interest in increasing the lowtemperature tolerance of Nile tilapia. Manipulation of environment, diet and genome is a potential method of improving low-temperature tolerance. This study evaluated the effects of selected environmental and dietary factors on survival and physiology when Nile tilapia were exposed to a slow (~ 0.5 8C day1) decrease in temperature. Although significant effects were observed in several cases, no treatment resulted in an increase in tolerance to low temperature that was considered to be of value to aquaculturists.

Acknowledgment This study was supported by the Aquaculture Research Initiative of the South Carolina Agricultural Experiment Station.

References
Al Amoudi M., El-Sayed A. & El-Ghobashy A. (1996) Effects of thermo-haline shocks on survival and osmotic concentrations of the tilapias Oreochromis mossambicus and Oreochromis aureus Oreochromis niloticus hybrids. Journal of the World Aquaculture Society 27, 456461. Allanson B.R., Bok A. & van Wyk N.I. (1971) The influence of exposure to low temperature on Tilapia mossambica Peters (Cichlidae). II. Changes in serum osmolarity, sodium and chloride ion concentrations. Journal of Fish Biology 3, 181185. AOAC (Association of Analytical Chemists) (1990) Official Methods of Analysis, 15th edn (ed. by K. Helrich). Association of Analytical Chemists, Arlington, VA. APHA (American Public Health Association), American Water Works Association and Water Pollution Control

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

249

Low-temperature tolerance of Nile tilapia H L Atwood et al. Ciereszko R.E., Dabrowski K., Ciereszko A., Eberling J. & Ottobre J.S. (1997) Effects of temperature and photoperiod on reproduction of female yellow perch Perca flavescens: plasma concentrations of steroid hormones, spontaneous and induced ovulation, and quality of eggs. Journal of the World Aquaculture Society 28, 344356. Cnaani A., Gall G.A.E. & Hulata G. (2000) Cold tolerance of tilapia species and hybrids. Aquaculture International 8, 289298. Craig S.R., Neill W.H. & Gatlin D.M. (1995) Effects of dietary lipid and environmental salinity on growth, body composition, and cold tolerance of juvenile red drum (Sciaenops ocellatus). Fish Physiology and Biochemistry 14, 4961. Craigie D.E. (1963) An effect of water hardness in the thermal resistance of the rainbow trout, Salmo gairdnerii Richardson. Canadian Journal of Zoology 41, 825830. Dan N.C. & Little D.C. (2000) Overwintering performance of Nile tilapia Oreochromis niloticus (L.) broodfish and seed at ambient temperatures in northern Vietnam. Aquaculture Research 31, 485493. Ellis A.E. (1990) Lysozyme assays. In: Techniques in Fish Immunology (ed. by J.S. Stolen, T.C. Fletcher, D.P. Anderson, B.S. Roberson & W.B. van Muiswinkel), pp. 101103. SOS Publications, Fair Haven, NJ. Farkas T., Csengeri I., Majoros F. & Olah J. (1980) Metabolism of fatty acids in fish. III. Combined effect of environmental temperature and diet on formation and deposition of fatty acids in the carp, Cyprinus carpio Linnaeus. Aquaculture 20, 2940. Fletcher T.C. & White A. (1976) The lysozyme of the plaice Pleuronectes platessa L. Comparative Biochemistry and Physiology 55B, 207210. Folch J., Lees M. & Stanley S. (1957) A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry 226, 497509. Hart P.R., Hutchinson W.G. & Purser G.J. (1996) Effects of photoperiod, temperature and salinity on hatcheryreared larvae of the greenback flounder (Rhombosolea tapirina Gunther, 1862). Aquaculture 144, 303311. Henderson R.J. & Tocher D.R. (1987) The lipid composition and biochemistry of freshwater fish. Progress in Lipid Research 26, 281347. Hrubec T.C., Robertson J.L. & Smith S.A. (1997) Effects of temperature on hematologic and serum biochemical profiles of hybrid striped bass (Morone chrysops Morone saxatilis). American Journal of Veterinary Research 58, 126130. Huang C., Huang M. & Hou P. (1998) Effect of dietary lipids on fatty acid composition and lipid peroxidation in sarcoplasmic reticulum of hybrid tilapia, Oreochromis niloticus O. Aureus. Comparative Biochemistry and Physiology 120B, 331336. Jennings D.P. (1991) Behavioral aspects of cold tolerance in blackchin tilapia, Sarotherodon melanotheron, at

Aquaculture Research, 2003, 34, 241251 different temperatures. Environmental Biology of Fishes 31, 185195. Jonassen T.M., Imsland A.K., Kadowaki S. & Stefansson S.O. (2000) Interaction of temperature and photoperiod on growth of Atlantic halibut Hippoglossus hippoglossus L. Aquaculture Research 31, 219227. Kelly A.M. & Kohler C.C. (1999) Cold tolerance and fatty acid composition of striped bass, white bass, and their hybrids. North American Journal of Aquaculture 61, 278285. Kindle K.R. & Whitmore D.H. (1986) Biochemical indicators of thermal stress. Tilapia aurea (Steindachner). Journal of Fish Biology 29, 243255. Lee R., Gerking S. & Jezierska B. (1983) Electrolyte balance and energy mobilization in acid-stressed rainbow trout, Salmo gairdneri, and their relation to reproductive success. Environmental Biology of Fishes 8, 115120. Mock A. & Peters G. (1990) Lysozyme activity in rainbow trout, Oncorhynchus mykiss (Walbaum), stressed by handling, transport and water pollution. Journal of Fish Biology 37, 873885. Muona M. & Soivio A. (1992) Changes in plasma lysozyme and blood leucocyte levels of hatchery-reared Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta L.) during parr-smolt transformation. Aquaculture 106, 7587. Parry R.M., Chandian R.C. & Shahani K.M. (1965) A rapid and sensitive assay of muramidase. Proceedings of the Society of Experimental Biology and Medicine 119, 384386. Peter R.E., Hontela A., Cook F. & Paulencu C.R. (1978) Daily cycles in serum cortisol levels in the goldfish: effects of photoperiod, temperature, and sexual condition. Canadian Journal of Zoology 56, 24432448. Popma T.J. & Lovshin L.L. (1996) World-Wide Prospects for Commercial Production of Tilapia. International Center for Aquaculture and Aquatic Environment, Department of Fisheries and Applied Aquaculture, Series no. 41. Auburn University, Auburn, AL. Popma T. & Masser M. (1999) Tilapia Life History and Biology. Southern Regional Aquaculture Center Publication no. 283. Ram R. & Singh R.S. (1988) Carbofuran-induced histopathological and biochemical changes in liver of the teleost fish, Channa punctatus (Boch). Ecotoxicology and Environmental Safety 16, 194198. Ridha M.T. & Cruz E.M. (2000) Effect of light intensity and photoperiod on Nile tilapia Oreochromis niloticus L. seed production. Aquaculture Research 31, 609617. Ridha M.T., Cruz E.M., Al-Ameeri A.A. & Alahmed A.A. (1998) Effects of controlling temperature and light duration on seed production in tilapia, Oreochromis spilurus (Gunther). Aquaculture Research 29, 403410. Satoh S., Poe W.E. & Wilson R.P. (1989) Effect of dietary n-3 fatty acids on weight gain and liver polar lipid fatty acid composition of fingerling channel catfish. Journal of Nutrition 119, 2328.

250

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

Aquaculture Research, 2003, 34, 241251 Seddon W.L. & Prosser G.L. (1997) Seasonal variations in the temperature acclimation response of the channel catfish, Ictalurus punctatus. Physiological Zoology 70, 3344. Seo C.W., Chui C.H. & Hodson R. (1994) Dietary effect on o3 fatty acid contents in hybrid striped bass. Journal of the American Oil Chemists Society 71, 661663. Sheldon W.M. & Blazer V.S. (1991) Influence of dietary lipid and temperature on bactericidal activity of channel catfish macrophages. Journal of Aquatic Animal Health 3, 8793. Smiley P.C. & Parsons G.R. (1997) Effects of photoperiod and temperature on swimming performance of white crappie. Transactions of the American Fisheries Society 126, 495499. Sovio A., Nyholm K. & Westman K. (1973) Notes on haematocrit determinations on rainbow trout, Salmo gairdneri. Aquaculture 2, 3135. Stauffer J.R. Jr (1986) Effects of salinity on preferred and lethal temperatures of Mozambique tilapia, Oreochromis mossambicus (Peters). Water Resources Bulletin 22, 205208. Stauffer J.R., Vann D.K. & Hocutt C.H. (1984) Effects of salinity on preferred and lethal temperatures of the blackchin tilapia, Sarotherodon melanotheron. Water Resource Bulletin 20, 771775. Strange R.J. (1980) Acclimation temperature influences cortisol and glucose concentrations in stressed channel catfish. Transactions of the American Fisheries Society 109, 298303. Strange R.J., Schreck C.B. & Golden J.T. (1977) Corticoid stress responses to handling and temperature in salmonids. Transactions of the American Fisheries Society 95, 372380. Sun L., Chen G. & Chang C. (1992) The physiological responses of tilapia exposed to low temperatures. Journal of Thermal Biology 17, 149153.

Low-temperature tolerance of Nile tilapia H L Atwood et al. Sun L., Chen G. & Chang C. (1994) Characteristics of blood parameters and gill Na-K-ATPase in chilled comatose tilapia cultured in various salinities. Comparative Biochemistry and Physiology 107A, 641646. Takeuchi T., Satoh S. & Watanabe T. (1983) Dietary lipids suitable for the practical feed of Tilapia nilotica. Bulletin of the Japanese Society of Scientific Fisheries 49, 13611365. Thomason J.C., Conn W., Le Comte E. & Davenport J. (1996) Effect of temperature and photoperiod on the growth of embryonic dogfish, Scyliorhinus canicula. Journal of Fish Biology 49, 739742. Tomasso A.O., Isely J.J. & Tomasso J.R. (1996) Physiological responses and mortality of striped bass angled in freshwater. Transactions of the American Fisheries Society 125, 321325. Umminger B.L. (1969) Physiological studies on supercooled killifish (Fundulus heteroclitus): serum inorganic constituents in relation to osmotic and ionic regulation at subzero temperatures. Journal of Experimental Zoology 172, 283302. Viola S., Mokady S., Behar D. & Cogan U. (1988) Effects of polyunsaturated fatty acids in feeds of tilapia and carp I. Body composition and fatty acid profiles at different environmental temperatures. Aquaculture 75, 127137. Woo N.Y.S., Ng T.B., Leung T.C. & Chow C.Y. (1997) Enhancement of growth of tilapia Oreochromis niloticus. iso-osmotic medium. Journal of Applied Ichthyology 13, 6771. Yashouv A. (1960) Effect of low temperatures on Tilapia nilotica and Tilapia galilaea. Bamidgeh 12, 6266. Zale A.V. & Gregory R.W. (1989) Effect of salinity on cold tolerance of juvenile blue tilapias. Transactions of the American Fisheries Society 118, 718720.

2003 Blackwell Publishing Ltd, Aquaculture Research, 34, 241251

251