Effect of sun-dried and fresh cassava leaves on growth of Tilapia (Oreochromis niloticus) fish fed basal diets of rice bran or rice bran mixed with cassava root meal

The growth response of Tilapia fish (Oreochromis niloticus) to supplementation with fresh or dried leaves of a sweet variety of cassava was studied in 1 m deep ponds (each 4 m * 2.5 m * 1 m). Rice bran or rice bran mixed with cassava root meal was provided as the energy source (20-24% of feed DM), the mixed feed being given at the rate of 5% of fish LW. There were 30 fish per pond with average initial weight of 6 g per head. The treatments were arranged as a 2*2 factorial in a Completely Randomized Design (CRD) with 4 replicates. The first factor was cassava leaf processing (sun-dried or fresh); the second factor was energy source (rice bran or rice bran mixed with cassava root meal). The cassava leaves and roots were from a local “sweet” variety traditionally planted by farmers for human consumption. The experiment was conducted for 100 days.

Daily gain in weight and length, and the ratio weight: length after 100 days of growth, did not differ among treatments. All the fish survived and there was no evidence of HCN toxicity. Water quality parameters were not affected by the treatments. There were no interactions between sources of protein and of energy.

It is concluded that the feeding of cassava leaves with rice bran can be readily adopted by small scale farmers as both these feed resources are readily available in rural areas.

Introduction

Poverty, population growth and environmental degradation (air, soil and water pollution) are increasingly being considered as focal points for research and development. The integration of livestock with trees, food crops and aquaculture is seen as the most appropriate way to use the natural resources in a system that is productive and sustainable (Preston 2000). Fish constitute more than 70% of the total animal protein intake of the Cambodian people and from 3.2 to 7.4% of the gross domestic product (GDP) (http://www.fao.org/fishery/countrysector/FI-CP_KH/en). However, fish caught from natural water surfaces such as lakes and ponds has declined from 120,000–130,000 tonnes in the 1960s (about 25kg per capita) to 75,000 tonnes in the 1990s (about 10-13 kg per capita). At the same time, the population of Cambodia continues to increase at an average of about 2.4%, reaching 14 million in 2009 (Censure-Cambodia 2008).

Aquaculture is now playing a significant role in the economy with production increasing from 1,610 tonnes in 1984 to 15,000 tonnes in 1999 (Nao Thouk 1999). This increase has been mainly with fish in cage and pond culture.

Recently, researchers in Cambodia have been focusing on developing ways to use protein-rich forages as a replacement for fish and soybean meals, especially for pig and rabbit production (http://www.mekarn.org/proprf/content.htm). Cassava leaves have been a focus for much of this research with pigs (Chhay Ty et al 2003a,b,c; Chhay Ty et al 2005a,b; Chhay Ty and Preston 2006; Chhay Ty et al 2009). Cassava leaves have been studied as protein sources for cattle (Ffoulkes and Preston 1978; Wanapat 2001), goats (Seng Sokerya 2009), and chickens (Khieu Borin 2005). However, their use as fish feed has not been studied at least in Cambodia.

The hypothesis underlying the present study was that both fresh and dry cassava leaves could be fed as a protein source to Tilapia fish (Picture 1) receiving basal diets of rice bran and rice bran with cassava root meal.

Materials and methods

Location

The experiment was carried out in the experimental farm of the Center for Livestock and Agriculture Development (CelAgrid) located in Preah Theat village, Rolous commune, Kandal Stung district, Kandal province, approximately 25 km from Phnom Penh city. The experiment started on 20^th August and finished on 28^th November 2009 (a total of 100 days).

Experimental design

The treatments arranged as a 2*2 factorial in a completely randomized design (CRD) with three replicates (Table 1) were:

Processing of cassava leaves

Energy source

Table 1. Experimental layout
Pond	1	2	3	4	5	6	7	8	9	10	11	12
	DCL-RB	FCL-RB-CRM	DCL-RB-CRM	FCL-RB	DCL-RB-CRM	FCL-RB	DCL-RB	FCL-RB-CRM	FCL-RB	FCL-RB-CRM	DCL-RB	DCL-RB-CRM

Pond construction and preparation

Twelve ponds were prepared each with a capacity of 10 m³ (4 m long, 2.5 m wide and 1 m deep). They were lined with blue plastic (polyethylene) sheet to protect them from soil erosion and to avoid filtration of water. Water from a large pond in the farm was pumped into all experimental ponds. Lime (CaO) at 200 g/m² was applied 20 days before stocking with fish in order to kill any parasites and pathogenic organisms and also to increase the pH (Pich Sophin and Preston 2001).

Experimental feeds

The cassava used in the experiment was a local (Damlong Kor) “sweet” variety considered to have a low level of HCN precursors. For the leaf and root meals, fresh leaves and roots were purchased from farmers who plant the cassava for human consumption. Leaves were sun-dried for 2-3 days and roots for 5-6 days, prior to grinding into a meal. The fresh cassava leaves were harvested in the experimental farm and were chopped in small pieces before feeding. The variety was the same as that purchased from the farmers. Rice bran was purchased from the local rice mill.

Feeding and management

The ingredients (Tables 2 and 3) were mixed together and offered three times per day at 7:00, 12:00 and 17.00h at the rate of 5% of fish live weight (DM basis).

Table 2. Composition of the diets, % DM basis
	DCL-RB	DCL-RB-CRM	FCL-RB	FCL-RB-CRM
Dried cassava leaves	80	83	0	0
Fresh cassava leaves	0	0	76	79
Rice bran	20	12	24	16
Cassava root meal	0	5	0	5
Total	100	100	100	100
% Crude protein	22.1	22.1	22.1	22.2

Experimental fish

A total of 360 Tilapia (Oreochromis niloticus) fingerlings were purchased 7 days after incubation from a local commercial hatchery. They were installed in the ponds (3 per 1 m²) and fed with commercial concentrate for one month before starting the experiment when the average weight was 6 g.

Measurements

Feeds

Samples of feed ingredient were analyzed for nitrogen, using a Foss-Tecator Kjeldahl apparatus (AOAC 1990), for DM by micro-wave radiation (Undersander et al 1993) and HCN determined by titrating with AgN0₃after boiling the sample in chloroform and reaction with 0.1N KOH (AOAC 1990).

Water quality parameters

Every 5 days, parameters of water quality were measured at a water depth of 20cm. Dissolved oxygen (DO) and pH were determined at 7.00am and at 2.00pm using a “Winkler” test-kit (Aqua Merck) (Picture 4) for the former and a glass electrode for the latter (Picture 5). Temperature was recorded at 07.00, 12.00 and 17.00h using the “Winkler” test kit. Water transparency was measured at 12.00 using a Sechii disk (Picture 6).

Fish growth rate

The weight and length (from the tip of the mouth to the caudal fin) of the fish were measured every 20 days in the morning at 7.00am. At the end of the experiment the total fish biomass was harvested and the weight and length recorded.

Statistical analysis

Daily increases in weight and length of the fish and the pH, DO, temperature and transparency were analyzed by the General Linear Model in the ANOVA software of Minitab version 13.31 (MTAB 2000).. The sources of variation were processing of cassava leaves, energy source, interaction between processing*energy and error.

Results and discussion

Toxicity

There was 100% survival of the fish on all treatments, indicating there was no hazard from giving the fresh cassava leaves to growing Tilapia. These findings are in agreement with the results of Du Thanh Hang and Preston (2005), Chhay Ty and Preston (2006) and Chhay Ty et al (2005a,b and 2009) in other non ruminant species (pigs) that health and production responses are not related with levels of ingestion of HCN precursors.

Growth in weight and length and in the ratio of weight:length

There were no treatment effects, and no interactions among treatments, for growth in weight and length, or the ratio of live weight to length (Tables 4 - 6; Figures 1-5).

Table 4. Mean values for changes in live weight of tilapia fish fed dry or fresh cassava leaves with basal diets of rice bran or rice bran mixed with cassava root meal.
Weight gain, g/day	Leaf processing			Energy sources
Weight gain, g/day	DCL	FCL	Prob	RB	RBCRM	Prob	SEM
0-20	0.26	0.36	0.08	0.33	0.29	0.52	0.04
20-40	0.20	0.35	0.15	0.31	0.23	0.48	0.07
40-60	0.58	0.63	0.79	0.66	0.56	0.65	0.14
60-80	0.72	0.71	0.95	0.78	0.66	0.59	0.16
80-100	0.76	0.98	0.25	0.85	0.88	0.87	0.12
0-100	0.50	0.59	0.33	0.58	0.51	0.45	0.06

Table 5. Gain in length of Tilapia fed dry or fresh cassava leaves with basal diets of rice bran or rice bran mixed with cassava root meal
Days	Leaf processing			Energy sources
Days	DCL	FCL	Prob	RB	RB-CRM	Prob	SEM
0-20	0.07	0.106	0.002	0.088	0.087	0.950	0.005
20-40	0.05	0.074	0.014	0.064	0.055	0.641	0.013
40-60	0.11	0.093	0.679	0.109	0.088	0.460	0.019
60-80	0.088	0.079	0.700	0.075	0.093	0.455	0.016
80-100	0.08	0.073	0.59	0.083	0.070	0.299	0.008
0-100	0.078	0.084	0.451	0.083	0.079	0.503	0.005

Table 6. Mean values for weight:length ratio of Tilapia fed dried or fresh cassava leaves with basal diets of rice bran or rice bran mixed with cassava root meal
Days	Leaf processing			Energy sources
Days	DCL	FCL	Prob	RB	RB-CRM	Prob	SEM
0	0.83	0.77	0.324	0.82	0.78	0.498	0.042
20	1.32	1.40	0.427	1.41	1.31	0.324	0.066
40	1.59	1.85	0.103	1.80	1.63	0.252	0.101
60	2.26	2.59	0.306	2.55	2.30	0.435	0.216
80	3.07	3.32	0.500	3.41	2.98	0.272	0.259
100	3.75	4.29	0.207	4.16	3.88	0.97	0.274

In general the mean values or growth in weight and length were towards the high end of those reported by other researchers (Table 7), who mostly employed only fertilization without feed supplements. In this latter case the fish are dependent on components of the natural food chain, such as plankton and other aquatic species. The advantage of the system employed in the present study is that the rice bran and cassava leaves are readily available in rural areas, which facilitates uptake of the technology by small scale farmers.

Table 7. Growth rates of Tilapia in ponds fertilized with pig manure or biodigester effluent, or not fertilized, with or without supplementation (reports from the literature compared with the present study)
Feed	Fertilizer	Wt gain, g/day	Length gain, cm/day	Authors
None	Biodigester effluent	0.27-0.43	0.041-0.044	San Thy et al and Preston 2003
None	Biodigester effluent	0.227	0.04	San Thy et al 2008
Water spinach	Biodigester effluent	0.516	0.068	San Thy et al 2008
Duckweed	Biodigester effluent	0.652	0.055	San Thy et al 2008
None	Biodigester effluent	0.499	0.041	Pich Sophin et al 2001
None	Pig manure	0.97	0.082	Nguyen Duy Quyinh Tram et al 2007
None	Biodigester effluent	0.72	0.073	Nguyen Duy Quyinh Tram et al 2007
Concentrate	Biodigester effluent	0.15-0.23g	-	Edwards et al 1988
Cassava leaves/rice bran	None	0.5 – 0.59	0.078 – 0.084	This study

Water quality

There were no differences among treatments in any of the measurements of water quality (Table 8), all of which were in the optimum range for growth of Tilapia, which are temperature 29 - 31°C (Popma and Lovshin 1995), pH 6.0 - 9.0 (Swingle 1969; Popma and Lovshin 1995); and DO concentration >5.0 mg/litre (Swingle 1969; Popma and Masser 1999; Popma and Lovshin 1995).

Table 8. Water quality parameter of Tilapia fed dried or fresh cassava leaves with basal diets of rice bran or rice bran mixed with cassava root meal
	Leaf processing			Energy sources
	DCL	FCL	Prob	RB	RB-CRM	Prob	SEM
Temperature, ⁰C
Morning	27.7	27.9	0.167	27.8	27.9	0.468	0.089
Afternoon	29.7	29.6	0.824	29.7	29.7	1.00	0.105
Evening	29.6	29.7	0.592	29.6	29.6	0.748	0.109
pH
Morning	7.48	7.50	0.434	7.50	7.47	0.233	0.019
Afternoon	7.49	7.48	0.784	7.49	7.48	0.969	0.02
Evening	7.45	7.46	0.505	7.47	7.46	0.506	0.026
Dissolved oxygen, mg/litre
Morning	4.99	5.04	0.931	4.94	5.08	0.799	0.392
Afternoon	5.38	5.47	0.861	5.42	5.43	0.974	0.351
Evening	6.36	4.78	0.254	4.84	6.30	0.289	0.976
Water transparency, cm
Afternoon	8.73	9.24	0.326	8.89	9.08	0.711	0.365

Conclusions

Acknowledgement

The Center for Livestock and Agriculture Development (CelAgrid) would like to express gratitude to the International Center for Tropical Agriculture (CIAT), which provided the fund for conducting this experiment.

References

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Table 3. Dry matter (DM), crude protein (CP) and HCN in the diets
	DM, %	CP, % in DM	HCN, mg/kg DM
Dry cassava leaves	91.2	25.3	60.0
Fresh cassava leaves	27.4	26.3	333
Rice bran	89.6	9.05	-
Cassava root meal	84.9	2.95	-