Growth performance and nutrient utilization of Clarias gariepinus fed with different dietary levels of processed cassava leaves

The growth performance of Clarias gariepinus fed with different dietary levels of processed cassava leaves and their nutrient utilized were studied for a period of 24 weeks. One hundred and forty four experimental Catfish (Clarias gariepinus) with 0.75±0.20 g mean weight and 3.9±0.31 cm mean total length were collected from Aqua-fish Consult, Awka, Anambra State, Nigeria. Eighteen rectangular plastic tanks were used for this study. Green cassava leaves (Manihot esculenta) were collected from a farm in Uda, Igbo-Eze North of Enugu State. The leaves were soaked for 36 h and sun-dried for one week to reduce the presence of cyanogenic glycosides. The dried leaves were ground into fine powder and analyzed for proximate composition. The experimental design used was Complete Randomized Block Design (CRBD) consisting of 18 treatments. Triplicate culture tanks in treated (L25-L100), and control groups (L0 and Ac) were stocked with eight fingerlings per square meter, respectively. The cost benefit of C. gariepinus fingerlings was estimated using weight gain and specific growth rate against management and construction cost. Weight gain, specific growth rate (SGR), feed efficiency (FE) and total length (TL) of catfish in different treatments were plotted against water chemistry parameters. This trail was conducted to access the possibility of replacing maize (Zea mays) with varying levels of whole cassava leaf meal in the diet of C. gariepinus catfish. Five isocaloric and isonitrogenous diets were formulated to contain 0, 25, 50, 75 and 100% cassava leaves to replace equal weight of maize meal. Internationally made fish feed (coppens) was also used as a control feed. All the diets were fed to catfish (C. gariepinus) fingerlings in replicate for 24 weeks. The results revealed that optimum requirement of cassava leaves level in the formulation of practical diets for improved growth of C. gariepinus was 25%.


INTRODUCTION
The overall effect of food insecurity is not only inadequate food production but also imbalances in the nutritional status of the populace at large (Aderemi et al.,2012).The dearth of animal products in the diet of an average Nigeria increases yearly, mainly due to poverty, high cost of animal feeds, political and economic instability coupled with decreased interest in animal production with greater efforts directed towards petroleum exploitation.Leaf meals of most tropical plants for example cassava leaf meal (Anyanwu, 2009) are available and cheap.Cassava is a multipurpose plant that thrives well in the tropics.It is a very good energy source widely grown in Nigeria.It has a wide range of adaptability, resistance to drought and tolerance to poor soils.
African Catfish (C.garepinus) are known to be omnivorous in their food habits (Anyarnwu et al., 2012).Besides, they are hardy and tolerant to a wide range of environmental conditions (Nwani et al., 2015).These attributes have indicated the fish as highly and veraciously disposed to accepting unconventional dietary feeds, such as leaf meals.The quest to intensify the culture of the fish so as to meet its ever increasing demand has made it vital to develop suitable diets either in supplementary forms in ponds or as whole feed in tanks (Olukunle, 2006).Feed is one of the major inputs in aquaculture production and fish feed technology has become one of the least development sectors of aquaculture particularly in Africa and other developing countries of the world (Gabriel et al., 2007).High cost of fish feed ingredients (maize and fish meal) is observed as one of the problems militating against aquaculture development in Nigeria (Gabriel et al., 2007).This leads to malnutrition of fish which subsequently results in decline in reproduction of individual fish.This eventually causes scarcity of fish species in the market which invariably results in high cost of fish.Leaf meals of most tropical plants, for example, cassava leaf meal (Anyanwu, 2009) are available and cheap.
Cassava roots and leaves are readily available and have also been found to support the growth of different fish species such as C. gariepinus (Catfish), Oreochromis niloticus (Tilapia), Mystus cavasinus (Catfish); but this can be limited by the presence of anti-nutrients such as linamarin.Linamarin is cyanogenic glycosides (2-B-Dglucopyranosy 1oxy-isobutryo nitrite) found in leaves and tuberous roots of cassava, which release high toxic cyanide (HCN) during hydrolysis at the time of digestion (Presston, 2004;Aderemi et al., 2012;Akapo et al., 2014).Previous researchers have attempted to increase non-conventional plant and animal materials to replace conventional feed ingredients like maize and fish meal in fish feed ration (Falaye, 1988;Fagbenro, 1992;Olatunde, 1996;Baruah et al., 2003;Eyo and Ezechie, 2004;Azaza et al., 2015).According to Olurin et al. (2006), maize is the major source of metabolizable energy in most compounded diets for Hetorebranchus x clarias hybrid species because it is readily available and digestible.Odo et al. 1185 However, the increasing prohibitive cost of this commodity has necessitated the need to search for an alternative source of energy.Few works are available on the replacement of maize with cassava root and cassava leaf in fish diet.These include those on mirror carp, Cyprinus Carpio (Ufodike and Matty, 1983), Rainbow trout, Salmo trutta (Ufodike and Matty, 1984), Tilapia, Oreochromis niloticus (Faturoti and Akinbote, 1986), Oreochromis mossambicus (Wee and Ng, 1986); catfish, C. gariepinus fingerlings (Olurin et al., 2006); C. gariepinus advance fry (Olukunle, 2006), C. gariepinus (Anyanwu et al., 2009(Anyanwu et al., , 2012) ) and Tinca tinca (Garcia et al., 2015); hence the quest to determine the growth performance and nutrient utilization of C. gariepinus species fed with varying dietary levels of cassava leaf meal as substitute for maize.

Procurement of C.gariepinus
One hundred and forty four experimental Catfish with 0.75±0.20 g mean weight and 3.9±0.31cm mean total length were collected from Aqua-fish Consult, Awka, Anambra State, Nigeria.After collection, they were transported to the wet laboratory of Zoology Department, University of Nigeria, Nsukka and allowed to acclimatize for 2 weeks with rectangular plastic tanks.

Preparation of culture tanks
Eighteen rectangular plastic tanks were used for this study for a period of twenty four weeks.Each tank has a water-holding capacity of 95 L, but water was maintained at 60 L level within the period of the research (Eyo, 1994).However, they were covered with mosquito net to prevent escape of fish, as well as entry of predators, and leaves from falling in.All tanks were continuously aerated using air pump.

Collection and processing of cassava leaves
Green cassava leaves (Manihot esculenta) were collected from a farm in Uda, Igbo-Eze North of Enugu State.The leaves were soaked for 36 h and sun-dried for 1 week to reduce the presence of cyanogenic glycosides.The dried leaves were grounded into fine powder and analyzed for proximate composition according to the procedure of APHA (2006).The proximate composition of the nutrient contents is shown in Table 1.

Formulation of fish feed with cassava leaves
Proximate analysis of dietary ingredients was carried out.Then, five isocaloric and nitrogenous diets were prepared: they contain 0, 25, 50, 75 and 100% processed cassava leaves, which were labeled L0, L25, L50, L75, and L100 respectively, to replace equal weight of maize.Prior to formulation of the five inorganic diets, experimental feeds were ground to a fine powder by using hammer mill machine.One kilogram of the feed was weighed out using a triple beam *Corresponding author.E-mail: gregory.odo@unn.edu.ng.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License The polythene bags were steamed for an hour.This was done to facilitate the gelatinization of starch and binding of diets.The diets were run through meat mincer fitted with 3mm dye and the resulting nodule shaped strands were cut into pellets and oven dried at 50°C for 3 h.The proximate analysis of the diet was carried out (AOAC, 1975) and shown in Table 2.
Control group Ac was fed with internationally made feed (coppens) while Control group L0 was fed with 0% dietary level cassava leaves.Prior to feeding, the fish in each tank was left for 2 days during which no artificial diet was administered to them, but was starved to allow utter digestion of any food in their stomach.At the end of acclimatization, fish in each tank were weighed (with mettler top loading balance) to determine their initial mean weight.Also mean total length and mean standard length measurements were taken using fish measuring board.The fish were fed daily in two rations (at 9:00am and 4:00pm) at a rate equivalent to 5% of the total body weight of the fish in each tank.The quantity of the diet administered was adjusted fortnightly using weight gain data.

Acceptability of diet
The acceptability of diet was accessed using the "time to strike index" (Eyo, 1994).The fish in every treatment was starved overnight to induce hunger.Pellet of diets for each treatment was dropped into the aquarium; the time which elapsed from the time the pellet penetrates the water and the moment the last fish struck the pellet with its mouth was recorded in seconds.The acceptability index was calculated as the reciprocal of the "time to strike".

Production parameters
All the fingerlings in each treatment were harvested and weighed collectively using mettler electronic balance (PC 2000) to the nearest 0.01g.

Weight gain
Weight gain was calculated using W2-W1, where W2 = final weight and W1= initial weight over a period.

Feed efficiency (FE)
Feed efficiency for catfish in different treatments was calculated using the formula; Where, feed intake (a) = feed eaten by the fish on a matter basis; weight gain (b) = a weight increase on wet matter basis (Boonyaratpalin, 1989).

Condition factor (K)
Condition factor of catfish in difference treatments was calculated using the formula: Where W = Weight of fish and L = total length of fish (Adikwu, 1992).

Total length (TL)
Total length of juvenile catfish in different treatments was recorded using fish measuring board to the nearest 0.01 cm.
Also, standard length of Juvenile catfish in different treatments was measured and recorded using fish measuring board to the nearest 0.01 cm.

Length-weight relationship (LWRs)
The length-weight relationship (LWRs) of the catfish in different treatments was plotted using the pooled data on length and weight of catfish in different treatments.

Water chemistry
Water quality parameters were measured during each sampling fortnightly.Temperature of the water was measured using mercuryin-glass thermometer, water pH was measured using Jenway P H metre, dissolved oxygen was determined using dissolved oxygen metre, total hardness mg/L, total ammonia mg/L and nitrite were measured using spectrometer.

Growth parameters-water chemistry relationship
Weight gain, SGR, FE and TL of catfish in different treatments were plotted against water chemistry parameters.The TW was used to calculate the Normalized Biomass Index (NBI) (Beck, 1979): Odo et al. 1187 Where, WF = Final weight of catfish in milligram, NF = Final number of catfish, W1 = Initial weight of catfish in milligram and N1 = Initial number of catfish.

Cost benefit analysis
The cost benefit of C. gariepinus fingerlings was estimated using weight gain and specific growth rate against management and construction cost.

Feed cost
The cost of feeding the fish was computed using (Lipton and Harmel, 2004): Where, Cfeed = cost contribution of feed to produce a pound of fish, P=per pound price of fish, WA = Weight added from purchase seed to harvest size (Harvest size -seed weight), FCR = Feed Conversion Ratio, and S=Percentage of fish surviving from seed to market size.

Seed cost
Cost of stocking at different densities was computed using: and Harmel, 2004).
Where, Cseed = Cost of contribution for producing a pound of fish, Pfeed= Purchase price of seed (Cassava leaves), W= Average weight of harvestable fish, S = Percentage of fish surviving from seed to market size.

Management cost
Management Cost was computed using (Lipton and Harmel, 2004): Where, Cseed = Cost of producing pound of fish; Cfeed = Cost contribution of feed to produce a pound of fish.All costs will be reduced to Naira (Nigeria National Currency) using Nsukka Urban Market Price.

Statistical analysis
Data resulting from the experiment were subjected to a two way analysis of variance using SPSS (Statistical package for social sciences) version 12. Turkey HSD, Dunkan, Fisher LSD and Tanhane were used to compare differences among individual means at P = 0.05.

Growth response
The total length of group Ac was the highest (12.85 ± 2.22a) while L100 had the lowest total length (10.65 ± 39 a ).The mean total length decreases in the following order: Ac > Lo > L 25 > L 75 > L 50 > L 100 (Table 4).
The mean weight of catfish fed diet L 100 (14.98 ± 4.61) was very low compared to those fed other diets.Also, diet L 25 had the highest mean weight (43.69 ± 17.51) among the varied levels of substitution of cassava leaf meal (Table 4 and Figure 1).

Weight gain
The mean weight of catfish fed diet L 100 (100% Cassava leaf meal) -14.98 ± 4.61 was very low compared to the mean weight of catfish fed diets L 0 ,L 25 , L 50 , L 75 and A c (41.17 ± 16.03, 43.69 ± 17.51, 32.45 ± 13.03, 26.85 ± 10.35 and 49.98 ± 19.43 respectively).Apart from group A c (Internationally made coppens fish feed) which mean weight is relatively high, that is, 49.98 ± 19.43, group L 25 had the highest mean weight gain (43.69 ± 17.51) among the varied levels of substitution of cassava leaf meal (Table 5).
Mean values with alphabets were compared with time to strike.Mean values with different alphabets differ significantly (P<0.05).Mean values with figures were compared with acceptability index.The time to strike differed significant (P<0.05) when diet groups were compared, with mean time to strike decreasing in the following order: L 100 <L 75 <L 50 <L 0 <L 25 <A C .The acceptability index of A C group was highest and it differed significantly (P<0.05) from those of the other groups.L 25 had the next high acceptability index followed by that of L 0 , L 50 , L 75 and L 100 .

DISCUSSION
The mean values for the water condition of the experimental aquaria (27.4± 0.55, 4.49± 1.23, and 7.1± 0.20) for temperature, pH and dissolved oxygen respectively fall within the optimal requirements for fish production (Ochang et al., 2007;Anyanwu et al., 2012).The highest mean feed efficiency was observed in diet Ac (coppens fish feed), followed by diet L 0 and then L 25 ; the lowest mean feed efficiency occurred in diet L 100.The relatively low feed efficiency recorded in diet L100 (100% CLM -9.82± 0.82) may be attributed to the presence of anti-nutrients in cassava leaf such as Linamarin (Presston, 2004;Azaza et al., 2015;Garcia et al., 2015).The chemical composition of cassava leaf meal showed very high level of crude fibre and low energy level, a future that is very common with leaf meals.The metabolizable energy value of the diets decreased with  increased levels of the leaf, indicating low energy status.
According to Lagler et al. (1977), the interaction of the factors affecting internal motivation or drive for feeding on specific diets range from intrinsic factor relating to physiology, genetics and morphology to extrinsic factors involving the living conditions especially food and feeding habits of the fish.Moreso, specific features such as size, age, sex, season and site of collection as well as species of fish, are of primary importance in determining the nutritional status and diet acceptability in catfish fed specific dietary types (Eyo, 1994).These factors however were taken care of since the catfishes were hatchery-raised, reared in the same pond and fed the same diet until they were collected for laboratory studies.
The incorporation of cassava leaf meal in the diet fed to C.gariepinus increased the palatability of the diets.The least mean time to strike (4.43± 0.08) was observed in diet Ac, followed by diet L 25 (5.42± 0.09).There was observed inconsistency in the mean-time to strike the graded levels of substitution of cassava leaf meal.Statistically, each diet was significantly different (p<0.05) from another.
Furthermore, despite the high acceptability observed in diet Ac. (Coppens fish feed), the acceptability index recorded in diet L 25 was significantly higher than those of other test diets.
The growth pattern revealed that C. gariepinus performed better in diet L 25 than all other diets.It has been documented that 50% replacement of maize with cassava root meal in broiler diet showed no depression in growth or unfavourable feed conversion ratio (Essers et al., 1995) and that the best growth performance was recorded in layers fed 10% cassava root meal.Olurin et al. (2006) reported a replacement level of 50% cassava meal for maize without a depression growth in C. gariepinus.In this present study, the best growth performance and nutrient utilization were recorded in fish fed 25% level of whole cassava leaf meal.This implies that the inclusion of 25% level of whole cassava leaf meal in the diet of C. gariepinus catfish enhanced growth rate.This is in line with the work of Ernesto et al. (2000;Amisah et al., 2009), where broiler had the best growth performance at 25% cassava root meal inclusion level.
However, the poor growth performance recorded in diet L 100 (100% CLM inclusion) may be attributed to the amount of anti-nutritional factors such as hydrogen cyanide (HCN) present in cassava leaf.This was indicated by the condition factor (k) of catfish in diet L 100 .In the present study, it is shown that the inclusion of cassava leaf meal at 75% and above led to poor growth performance of the catfish.Feed stuffs which have antinutritional factors recorded poor growth performance in fish when supplemented at high levels (Ugwu and Mgbenka, 2006;Adewolu, 2008).Replacement of corn meal with cassava leaf meal recorded the highest mean weight gain and specific growth rate among catfishes fed diet L 25 (25% CLM inclusion).This represents the highest level of CLM incorporation.On the contrary, the least mean weight gain and specific growth rate were recorded in diet L 100 (100% CLM inclusion).
Furthermore, the ability of an organism to convert nutrients especially protein positively influences its growth performance.This was justified by the growth performance in 25% whole cassava leaf meal inclusion diet.Lower feed conversion ratio indicates better utilization of the feed by the fish.According to De Silva and Anderson (2001), feed conversion ratio is between 1.2 to 1.8 for fish fed carefully prepared diets, and the results from the present study fall within this range when multiplied by 10.The least mean feed conversion ratio was observed in diet L 25 , indicating that fish in this diet had the best utilization of the feed.However, the mean feed conversion ratio was highest in diet L 100 (100% CLM inclusion), indicating that fish in this diet had the worst utilization of the feed.Diet L 100 was significantly different (p<0.05) from all other diets.
In addition, condition factor (k) of fish in diet Ac (internationally made coppens fish feed) and diet L 25 (25% CLM inclusion) were not significantly different (p>0.05).Both had the best condition factor, implying that 25% CLM inclusion is the best rate of CLM substitution for maize in the growth of C. gariepinus catfish (Azaza et al., 2015).The optimum survival rates recorded in this study indicate that feeding C. gariepinus catfish with processed cassava leaf meal does not lead to mortality of the fish.This may probably be due to the substantial reduction in the cyanide content (by boiling and drying) of the whole cassava leaf meal (Garcia et al., 2015).Cardoso et al. (2005) observed that good processing of cassava enhanced survival and healthy state of fish at all stages of their lives.

Conclusion
Based on the results obtained in this study, it is recommended that under standard culture conditions, up Odo et al. 1191 to 25% cassava leaf meal can be used to substitute maize.Cassava leaf is abundant in Nigeria and extremely less expensive compared to maize.This will reduce overdependence on imported fish feed and maize feed used in aquaculture.

Figure 1 .
Figure 1.Weight gain of Clarias gariepinus juveniles fed different dietary levels of cassava leaf meal substitution for 24 weeks.

Figure 2 .
Figure 2. Time to strike and acceptability.

Table 1 .
Proximate composition of nutrient content of cassava leaf.

Table 2 .
Percentage composition of experimental diets in different treatment (cassava leaves inclusion) .

Table 3 .
Acceptibility index of C. gariepinus fed different dietary levels of CLM.

Table 4 .
Total Length (cm) of C. gariepinus juveniles fed graded levels of cassava leaf meal (CLM) substitution for 24 weeks.

Table 5 .
Growth performance and nutrient utilization of Clarias gariepinus fed different dietary levels of cassava leaf meal substitution for 24 weeks.

Table 6 .
Water quality fluctuations during the 24 week feeding trial of C. gariepinus juveniles fed graded levels of CLM substitution.