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Many aquaculture species are currently exposed to increasing water temperature and the general trend is predicted to continue (Pittock, 2003; Lorentzen, 2008; Lough and Hobday, 2011; FAO/NACA, 2012). Salmonid culture in Australia is approaching to the upper thermal tolerance limit for species (Pankhurst and King, 2010; Barnes et al., 2011; Hobday and Lough, 2011; De Silva, 2012). All cultured aquatic species are poikilothermic and increasing water temperatures cause an exponential increase in metabolic rate, this combined with decreasing oxygen solubility result in negative effects on growth performance (Jobling, 1997; Katersky et al., 2006; Barnes et al., 2011). Since fish consume to meet their energy requirement, feeding fish at elevated temperatures to satiation means fish will consume enough food to meet their metabolic demand, feed intake will increase until appetite is inhibited (Kaushik and Médale, 1994;

174 Jobling, 1997; Battaglene et al., 2008). When ration is restricted, the scope of growth is progressively reduced at high temperatures because more energy is allocated for maintenance metabolism (Jobling, 1994). Satiation feeding at high temperature is assumed to be beneficial; however, unlimited supply of food is not practical to improve the growth and feed efficiency at high temperatures due to the effects of increased oxygen demand with feeding (Jobling, 1994; Jobling, 1997; Katersky et al., 2006). The benefit of satiation feeding diminished at high temperatures in coral-reef fish,

Acanthochromis polyacanthus and growth rate was not significantly increased compared to low temperatures (Munday et al., 2008). The growth and feed efficiency of brook trout was lower at 19°C than at 15°C which is consistent to Atlantic salmon, where growth rate and feed conversion efficiency (FCE) was lower at 18°C than at 14°C (Chapter 2, 4, 5; Handeland et al., 2008).

Within the thermal tolerance range, metabolic rate (energy losses) is increased with temperature and nutrient ingestion rate (energy intake) is also increased and peak at the optimal temperature (Jobling, 1997). The difference between the rate of energy intake and the rate of energy loss represents the resources available for growth (Jobling, 1994; Jobling, 1997). In addition, high temperatures reduce dissolve oxygen (DO) level, which limit the growth potential at high temperature due to as inability of the respiratory system to provide oxygen to respiring tissues under high oxygen demand for increased metabolism (Jobling, 1997; Katersky and Carter, 2007a). The oxygen demand (O2·fish-1·h-1) of fish is increased during and shortly after feeding, known as specific

dynamic action (SDA), which is approximately double the routine metabolism and remains elevated for long periods of time, depending on water temperature, meal size and composition (Jobling, 1994; Carter and Houlihan, 2001; Katersky et al., 2006). Consequently, increased SDA accelerates the stress of fish at high temperature and limits the ability of fish to consume feed (Katersky and Carter, 2007a) and this may be the cause of lowered feed intake of brook trout at high temperature in this study. Energy required for increased SDA is also obtained from digestion and absorption of nutrient and protein catabolism (Jobling, 1994). The lower growth potential of fish at high temperature is a cumulative effect of increased energy expenditure for increased metabolism, reduced feed intake and decreased oxygen availability (Jobling, 1997; Koskela et al., 1997; Katersky and Carter, 2007a).

175 Since the more nutrients are catabolised for increased metabolism at high temperature, the whole-body chemical composition of fish is affected (Jobling, 1994; Koskela et al., 1997; Bendiksen et al., 2003; Katersky and Carter, 2005). When fish are fed, they grow and the supplied nutrients are deposited in the body as protein and lipid. The rate of protein deposition is the difference between the rate of protein synthesis and the rate of protein degradation, is termed as protein turnover (Jobling, 1994). Protein turnover occurs when protein synthesis exceeds protein degradation and it is evident that the rate of protein synthesis is maximised at the optimum temperature of any fish species (McCafferty and Houlihan, 1997). Higher levels of whole-body protein at high temperature was found carp Labeo rohita (Kumar et al., 2012), however, when temperature exceeded the optimal temperature whole-body protein level is decreased (Katersky and Carter, 2005). The lower whole-body protein in brook trout at 19°C compared to 15°C was possibly due to lower protein turnover and increased metabolism at high temperature (Jobling, 1994; Katersky and Carter, 2005; Katersky and Carter, 2007b). Whole-body lipid in brook trout was higher at 15°C than at 19°C and was similar to previous studies where whole-body lipid levels of fish peaked at the optimal temperature and then decreased at higher temperatures (Koskela et al., 1997; Katersky and Carter, 2005). A similar trend in whole-body energy content of brook trout was also evident as seen in previous studies (Koskela et al., 1997; Katersky and Carter, 2005), was likely a result of increased energy demand at high temperature (Jobling, 1997). Brook trout required more energy at high temperature (chapter 5).

Protein and lipid are used as energy sources in carnivorous fish, which are obtained from fish meal and fish oil, respectively. Expansion of the aquaculture industry in areas where temperatures approach the upper thermal tolerance level for a given species means an increasing demand of wild harvested fish to provide protein and oil (Hobday et al., 2008). In spite of the increasing demand, the production of fish meal and fish oil remains static; and their price has increased annually (FAO, 2012). In order to produce cost effective aquafeeds at elevated temperatures, alternative energy sources from either plant protein and oil or carbohydrate need to be added and balanced.

In all experiments in this study, nutrient digestibility, including carbohydrate energy was lower at high temperature. Lower apparent nutrient digestibility at higher temperatures in brook trout may be caused by faster gastric evacuation rates (He and

176 Wurtsbaugh, 1993; Sweka et al., 2004; Pérez-Casanova et al., 2009). Temperature accounted for 72-91% of the variation of gastric evacuation rate in fish (Garcia and Adelman, 1985). Gastric evacuation rate of brook trout was estimated at 4°C to 17°C and it was increased with increasing temperature (Sweka et al., 2004). However, in this study when the dietary lipid level decreased, the digestibility of carbohydrate energy increased at both temperatures (15°C or 19°C) (Chapter 4). Higher levels of dietary lipid had a negative effect on nutrient digestibility as well as carbohydrate digestibility in Atlantic halibut (Berge and Storebakken, 1991) and ruminant animals (Doreau and Chilliard, 1997). Considering these findings, aquafeeds with higher carbohydrate energy rather than lipid energy may be a better choice of energy source at high temperature.

Some enzymes associated with energy production from carbohydrate show temperature compensation (Couto et al., 2008; Enes et al., 2008b; Moreira et al., 2008). The breakdown of dietary carbohydrate for energy production means carbohydrate as glucose is converted to pyruvate through glycolysis and subsequently it is broken down to energy through Krebs cycle (Rawles et al., 2008). The activity of enzymes involved with glycolysis were increased with increasing temperature in fish indicated that carbohydrate was used as an energy source to fulfil the energy requirement (Hilton et al., 1982; Couto et al., 2008; Enes et al., 2008b; Enes et al., 2008a). Pyruvate kinase activity was higher at elevated temperature indicating that carbohydrate utilisation was higher at 19°C than 15°C (Chapter 3). This was also evident in rainbow trout where more dietary carbohydrate was utilised for energy at 18°C in comparison to 8°C (Brauge et al., 1995).