The effect of temperature on the physiological condition and growth performance of freshwater eel elver Anguilla bicolor bicolor ( McClelland , 1844 )

This study aimed to analyze the effect of water temperature on the physiological condition and growth performance of freshwater eel elver Anguilla bicolor bicolor (McClelland, 1844). This study was conducted in March 2017 at the Physiology Laboratory of Aquatic Animal, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University. The study used a completely randomized design with five different levels of temperature (22°C, 24°C, 26°C, 28°C, and 30°C) as treatments with two replications. The size of elver was 2–3 g. The experimental fish were fed with 1 mm pellet containing 45% of protein. The feeding level was 7% of fish biomass and the feeding frequency was two times a day. The results showed that temperatures range from 24–30°C could be used for freshwater eel elver rearing and 28–30°C were the best temperatures to support survival and growth performance of eel elver. A temperature of 24°C was the best temperature that could reduce the metabolism rate and did not cause stress on the elver.


INTRODUCTION
Freshwater eel Anguilla spp.has a high economical value and one of the export commodities in the fisheries sector.The demand for freshwater eel is very high and it keeps increasing up to 600.000 tons in 2012 (Sugeha, 2013).The prices of freshwater eel ranged between Rp 180.000 to Rp 300.000 per kg.However, the demand for freshwater eel was not in line with the supply.The massive aquaculture activity, especially in East Asia countries (Japan, Taiwan, and China) and in European countries (Italy and Germany) caused massive exploitation of glass eel and elver in the nature (Arai, 2016), therefore the population of glass eel and elver have been decreased drastically (Arai, 2014;ICES, 2011;Solomon & Ahmed, 2017).The supply of glass eel and elver were decreased up to 80% (Japan) and 99% (Europe) from 1984 to 2000 (Dekker et al., 2003).In order to compensate for market demand and availability of stocks in nature, it is necessary to conserve the eel resources in nature, by adding stocks in natural waters through the restocking of eel seeds and avoiding catching seeds from nature.
The important factor that affects the survival and growth of fish instead of their feed is the water quality especially the water temperature.Effendi (2003) stated that water temperature is an important factor in the metabolism process, then it affects the fish survival and growth and fish reproduction.The changes in water temperature in sudden or the occurred of extreme temperature will negatively affect the fish survival, even further cause mass mortality.The decrease of temperature followed by the decrease of enzyme activities in body metabolism could reduce the appetite, therefore as the decreasing of the consumed feed, the growth rate will decrease as well.Syahailatua (2008) and Nerici et al. (2012) reported that the increasing of water temperature will be followed by the increasing of metabolism degree to fulfill the oxygen need of the organism.This was in line with Van't Hoff rule which stated that the velocity of chemical reactions is increased by two folds or more for each 10°C in temperature rise.Deelder (1984) proposed that European eel A. anguilla can adapt to the water temperature at a range from 0 to 30°C.The feeding activity in A. anguilla was stopped in less than 10°C and more than 38°C (Sadler, 1979) and less than 10°C and more than 36°C in A. japonica (Son et al., 2011).Tesch (2003) has shown that the optimum of water temperature for rearing of A. japonica elver is at a range of 24-28°C and 23-26°C for A. anguilla.The optimum temperature for the rearing of A. rostrata glass eel is 18-22°C (Blakeslee et al., 2017).According to that, the aim of this study was to analyze the effect of water temperature on the physiological condition and growth performance of freshwater eel elver Anguilla bicolor bicolor (McClelland, 1844).The study used a completely randomized design with five different temperature treatments (22, 24, 26, 28, and 30±1°C) with two replications.The feeding rate was 7% of the fish biomass for rearing within a month.The procedure of this study was described below: was obtained from PT. Laju Banyu Semesta.That powder feed than was solidified into 1 mm of sinking pellet.

MATERIALS AND METHODS
Prior to experiment, the elver was acclimatized for a week.The elver was reared with different temperature treatments for 30 days.The elver was stocked as many as 200 fishes with the stocking density of 20 fishes/aquarium and fed twice a day at 08.00 am (25% from total feed) and at 04.00 pm (75% from total feed).The fish fed every day continuously with the feeding level about 7% of fish biomass.In every morning before feeding, the rearing media was siphoned to remove the residual feed.Every aquarium was equipped with a heater set at the expected temperature and the max-min thermometer to control the temperature.The rearing was conducted in a room with an air conditioner with a temperature of 20°C.

The observation and measurement procedure
The parameters that observed in this study were fish mortality and the physiological condition of the fish.The observation was done every day during the rearing period according to the treatments.The parameters that measured in this study were the measurements of body length (0.1 mm) and body weight (with 0.01 g of accuracy).The measurement was done four times every seven days during the experiment.
The blood glucose was measured using GOD-PAP (glucose oxidase phenol 4-aminoantipirin) (Sacks et al., 2011), haematocrit, andhaemoglobin (Practice Guidelines, 2015).The blood test was done at the initial and at the end of the experiment.The measurement of chemical composition in elver was done by Weende Experiment Station method (CCP, 2015).The measurement of oxygen consumption rate was performed at the end of the study by closed-loop measurement system (Novita et al., 2011).

The tested parameter
The tested parameters were physiological condition, growth performance, and water quality.The physiological conditions were body glucose, haematocrit, haemoglobin, and oxygen consumption rate.The growth performance parameters were survival (SR), specific growth rate (SGR), a weight coefficient of variance (CV), feed conversion ratio, and chemical composition of the body.Meanwhile, the water quality parameters were temperature, dissolved oxygen, pH, and ammonia.
The measurement of the growth parameter was done once a week, whereas the physiological condition (stress response) was performed at the initial and the end of the study.The measurement of temperature was conducted every day, while the dissolved oxygen, pH, and ammonia was carried out once in a week, and the measurement of oxygen consumption rate was done at the end of the study.All the parameters were calculated by the following formula below:

Data analysis
The obtained data in this study were analyzed by using Microsoft Excel 2010 and SPSS 21.1 including the homogeneity, normality, analysis of variance (ANOVA) and F test in 95% of confidence level.This analysis was used to determine the significant differences among treatments toward tested parameter such as survival rate (SR), specific growth rate (SGR), a weight coefficient of variance (CV), and feed conversion (FC).If the data is significantly different, the test will continue to post hoc test in 95% of confidence level (Steel & Torrie, 1981) to obtain the difference within treatment.The descriptive analysis was used to explain the effect of the treatments toward glucose value, haemoglobin, haematocrit, oxygen consumption level, the chemical composition of the body, and water quality during elver rearing.

Physiological condition
The blood characteristic of elver during the rearing is shown in Table 1.In 24°C, the data of the glucose level, haemoglobin, and haematocrit were the closest value related to blood characteristic in elver before the treatments.
Oxygen consumption level was increase (0.20-0.41 mg O2/g/hour) along with the increase of temperature (22, 24, 26, 28 and 30°C).Blood glucose, haemoglobin, and haematocrit experienced a fluctuation, yet they were still in normal range.

Growth performance
The growth performance of the elver during the rearing is shown in Table 2.The average of the fish survival was more than 85% in 24-30°C.The increase of temperature was followed by the increase of specific growth rate and a weight coefficient of variance.However, the feed conversion was decreased along with the increasing of rearing media temperature.

The body chemical composition of elver
The result of the proximate analysis of elver reared in different temperatures (22 °C, 24 °C, 26 °C, 28 °C, and 30 °C) is displayed in Table 3.The results showed that there was a different body chemical composition of elver in every treatment.

Water quality
The average value of the physical and chemical of water quality parameters in the elver rearing media with different temperature treatments during the rearing period is presented in Table 4.
All the average value of physical-chemical parameters during the rearing period was still in the optimum average for elver.Generally, the increasing temperature in the rearing media was followed by the increase of pH and ammonia.

Discussion
Eel Anguilla bicolor bicolor has a wide temperature tolerance.Blakeslee et al. (2017) found that the optimum temperature for glass eel A. rostrate rearing was 18 to 22°C.In this recent study, the elver reared at temperature of 24-30°C have showed normal condition, yet the elver started to stress in 22°C media temperature.This condition was displayed in the blood characteristic (i.e.glucose, haemoglobin, and haematorit) that started to show differences from that of the normal condition and the oxygen consumption level was low (0.20 mg O2/g/hour) and the elver was less active during the rearing in temperature of 22°C.Porchas et al. (2009) stated that the blood glucose can be used as the stress indicator of fish and the blood glucose concentration can express the health status of fish (Bartoňková et al., 2016).
During the elver rearing, the blood glucose was 38.54-65.45mg/dL.Rahardjo et al. (2011) stated that the normal blood glucose level in fish is 40-90 mg/dL.This value is relatively close to normal blood glucose level in human, i.e. 70-110 mg/dL.The blood glucose is an important component for many functions in some specific tissues, including the brain, the gill, the erythrocyte, and the gonads (Polakof et al., 2012).Blood glucose in fish body is the main energy source, fuel supply, and essential substrate for cell metabolism, especially for brain cells metabolism.Fish has a mechanism to maintain the blood glucose in several stages (Soengas, 2014) and has the different blood glucose level depend on the fish size and activity (Driedzic et al., 2013).Yasemi and Nikoo (2010) declared that in stress condition, the glucocorticoid level increases resulted in the increase of blood glucose level.This condition is used to overcome the high needs of the energy during stress.That condition can be fulfilled if only the blood glucose is absorbed into cells immediately.The glucose in cells will be catabolized soon to fulfill the needs of fish body physiology and energy.After the needs are fulfilled, the glycogenesis and lipogenesis process will be started (Polakof et al., 2012;Kamalam et al., 2016).
The haemoglobin (Hb) value of elver after rearing showed 3.36-6.27per 100g/mL.This result showed an insignificant increase compared with the control and the haemoglobin value after rearing was still within a normal range.This result was in accordance with Zuhrawati (2014) who reported that there was no significant effect of temperature to haemoglobin level in Oreochromis niloticus.Hb is a protein as a key in transporting the blood oxygen in fish (Rummer & Brauner, 2015).Radoslav et al. (2013) have shown that the increasing of haemoglobin reflects a stress condition of the fish.Physiologically, the haemoglobin determines the immune system level of fish because its strong connection with the blood.Moreover, the blood ability that used to carry the oxygen is relied on the Hb level (Rummer & Brauner, 2011).
Haematocrit or packed cell volume (PVC) is erythrocyte volume in 100 mL of blood or the comparison within the erythrocyte and the blood plasma in percent (%).The haematocrit of elver during the rearing was between 8.58-20.74%.The result showed that the haematocrit value was increase compared with the control.Even the blood haematocrit of elver after the rearing was increase but insignificantly different with the haematocrit before the treatments.The haematocrit level after the rearing was still in normal condition.The increasing value of hematocrite level were related with increasing of temperature (Bozorgnia et al., 2011).
The oxygen consumption level is one parameter to understand the fish metabolism rate and the oxygen consumption level is affected by the water temperature (Remen et al., 2015).Table 2 showed that the increasing of water temperature (22-30°C) was followed by the increasing of oxygen consumption level in elver.When the temperature was increase, the oxygen-utilizing also increase (0.05 mgO2/g/hour) with the oxygen content reached a range of 5-6 mg/L.The increase of temperature as much as 1°C would increase the oxygen consumption level to 10% (Boyd, 1988).At 30°C media temperature, the oxygen consumption level of elver was 0.41 mg O2/g/hour.This finding was in line with the study of Saputra et al. (2016) which used elver with a size of 3 g/fish at a temperature ranged from 29 to 31°C.Generally, metabolism rate per weight in smaller fish was higher than that of the bigger one.Therefore, smaller fish required more oxygen than the bigger one.The relationship between the metabolism rate and fish body size was affected by some intrinsic factors (i.e.physiology) and extrinsic factors (i.e.environmental parameter) (Urbina & Glover, 2013).Scabra et al. (2016) reported that the oxygen consumption level of elver with size of 10 g/ind was 0.36 mg O2/g/ hour.KKP (2011) has shown that the eel needs an environment with the oxygen demand of more than 4 mg/L.The lack of oxygen in rearing media can lead to fish stress and mortality (Diaz & Breitburg, 2009).
Even the result of analysis of variance showed that the temperature treatments (22°C, 24°C, 26°C, 28°C, and 30°C) in rearing media were significantly affect the survival rate, but elver was strong enough to deal with the temperature changes.The research showed that survival rate of elver was high.The survival rate of elver was more than 90% in 24-30°C temperatures, while the weak condition of elver with the survival of 80% was observed in media temperature less than 24°C.The temperature treatments ranged of 24 to 30°C are still tolerated by the elver.
Metabolism rate increases when temperature increases (Schulte, 2015).Thus, the need of energy and consumption rate are raise (O'Connor, 2009;Lemoine et al., 2013).Consequently, the growth rate of elver was increase along with the increase of temperature.Our study showed that there was a difference in coefficient of variance of body weight in the different temperature treatments.In the low temperature treatment, elver showed a lower appetite and less movement, therefore the competition in getting feed was low.Accordingly, the low variation of body size was observed in line with the decreasing water temperature, otherwise body weight coefficient of variance was high along with the increasing of the water temperature.
The feed conversion value was fluctuated in accordance with temperature treatments (22°C, 24°C, 26°C, 28°C, and 30°C).There was a tendency that FCR value decreases when the temperature increases.The feed conversion ratio (FCR) defines the feed requirement (in kg) to obtain 1 kg of body weight.The feed conversion value is inverse with the feed efficiency value, the lower the FCR value, the higher the feed efficiency is.
Overall, the physico-chemical parameters showed an optimum media condition for elver.The water temperature affects the metabolism rate, feeding activity (Maltez et al., 2016), and the growth of elver (Lefebvre et al., 2011).The increasing temperature will be followed by the increasing of metabolic rate (Claësson et al., 2016).The water temperature affects the oxygen solubility in water.The solubility of oxygen decreases as temperature increases (Kale, 2016).The ammonia toxicity will increase along with the increase of pH and temperature (Effendi, 2003).The best temperature for the physiological growth of eel as shown in the fish survival was 24-30°C.The pH value for elver rearing was ranged from 7.05-7.98.EFSA (2009) stated that the best pH for eel rearing is neutral with pH ranged from 6.0-8.0.Bhatnagar and Devi (2013) explained that to increase the productivity, the dissolve oxygen in water should be 5 mg/L.The increasing water temperature will affect the fish physiology (Claësson et al., 2016).The increasing of temperature in the optimum range was followed by the feeding activity and higher needs of oxygen.This caused the oxygen consumption level becomes higher and the ammonia content as excretion outcome increases (Mendonça & Gamperl, 2010;Healy & Schulte, 2012;Clark et al., 2013;Gräns et al., 2014;Norin et al., 2014).Ammonia in water can enter the blood stream of the elver through the gills, thereby it causes methemoglobin (brown blood disease), that can kill the fish because the fish experienced hypoxia (the lack of oxygen supply) (Wiesman et al., 2007).During the research, the ammonia content is still tolerable by elver.Yudiarto et al. (2012) found that ammonia concentration between 1.5-2.0mg/L still can be tolerated by the elver.

CONCLUSION
The optimal temperature to support the survival and the growth of elver is between 28-30°C.The temperature of 24°C is the recommended temperature to maintain low metabolism rate without causing stress in elver.

(
growth rate (% /day) Wt = Elver biomass at the end of the study (g) Wo = Elver biomass in the initiation of the study (g) t = Time of rearing (day) of elver at the end of the study (g) Bo = Total biomass of elver in the initiation of the study (g) Bm = Total biomass of dead elver during the study (g) F = Total feed (g)

Table 1 .
Blood characteristic of elver Anguilla bicolor bicolor during the rearing period Note: Mean value followed by the same superscript shows significant differences in 95% of confidence level (post hoc test)

Table 2 .
The growth performance of the elver during the rearing period

Table 3 .
Proximate analysis of elver's body (% wet weight) during the rearing period

Table 4 .
The average value of the physical-chemical composition of water during the rearing period Quality in Warmwater Fish Ponds.Fourth Printing.Alabama (US): Auburn University Agricultural Experiment Station.Bozorgnia A, Alimohammadi R, Hosseinifard R. 2011.Acute Effects of Different Temperature in the Blood Parameters of Common Carp (Cyprinus carpio). 2 nd International Conference on Environmental Science and Technology IPCBEE.Vol.6.IACSIT Press, Singapore.[CCP] Cereals and Cereal Products.2015.Manual for analysis of cereal and cereal products.India (ND): Ministry of Health and Family Welfare.Claësson D, Wang T, Malte H. 2016.