Replacing earthworm by water spinach or duckweed on growing frogs with basal diet of rice bran and broken rice 

Chhay Ty, Ung Ratha, Vor Sina,  Khieu Borin and T R Preston^* 

Center for Livestock and Agriculture Development (CelAgrid). Pras Teat village, Rolous commune, Kandal Stung district, Kandal province.  PO Box 2423 Phnom Penh 3, Cambodia
chhayty@celagrid.org
* Fundación para la Producción Agropecuaria Tropical Sostenible Capitulo Colombia

Abstract

The aim of the experiment was to study on replacing earthworm by water spinach or duckweed on growing frogs with basal diet of rice bran and broken rice in 18 ponds with area of 2 m² (2m x 1m) and 0.4m deep in each pond. All ponds were lined with white plastic in order to avoid filtration of water. A total of 144 frogs were bought from farmer and adapted to experimental area and feed for 10-15 days before started experiment. Frog stocking densities were 4 frogs per m². The experiment design was Completely Randomized Design (CRD) with 3*2 factorial arrangements of 6 treatments and three replications within each treatment, the first factor was protein sources from earthworm (EW) alone, duckweed mix with earthworm (DW-EW) or water spinach mix with earthworm(WS-EW) and second factor was with or without light supplementation at night. In overall, the DM feed offer and CP offer were not different of DW-EW and EW but lowest was found in WS-EW treatment (P<0.001). DM feed offer and CP offer were high when light was supplement rather than without light supplementation. The live weight gain were found the best in all period of experiments when the frogs fed EW diet rather than WS-EW or DW-EW treatment (P<0.05). Supplementing light was effect on live weight gain compared with without light supplementation (P<0.05) but accepted from 30-60 days (P>0.05). The feed conversion ratio in DM basis was better on DW-EW and EW alone and poorest was WS-EW treatment (P<0.001), while the light supplementation was not effect (P>0.05) but excepted period of 60-90 days, feed conversion ratio were better on light supplementation (P<0.05). The survival rate and net yields were highest on earthworm treatment and not different in mixture of DW-EW and WS-EW. Light supplementation was effect on high survival rate and yield compared with without light supplementation. It can concluded that growth rates, survival and yield were best on treatment earthworm alone and light supplementation, while the mixture of duckweed with earthworm were improved but poorest was water spinach mix with earthworm.

Key words: earthworm, water spinach, duckweed, light, live weight, survival, yield 

Introduction

Frog meat are important for human consumption and it is more economic in some countries such as France, Japan, Germany, Singapore, Hong Kong, Singapore and Malaysia (Akasay, 1994). In Cambodia, Many consumers are preferring frog meat, event in restaurant; they preserved frog food because frog meat can make much kind of foods and good taste and most of the frog come from the natural. However, in Cambodia, the cultivation of frogs is still not attractive because of farmers don’t know how to rise or how to fed to that animal. In recently, Care international in Cambodia has introduce frog raising to farmer as pilot activities in Pailin province but farmers fed mix concentrate feed with rice bran which is not economic retune. However, there are other alternative feed which farmers can use as protein sources such as earth worm, water spinach, duckweed and energy sources come from by product such as rice bran, broken rice etc.

Earthworms are appropriate elements in systems of recycling livestock manure and have high in protein (59.9%), dry matter (15-20%), crude fiber (7.43%), Ca (1.73%) and P (0.118%) (Bay, 2002). Earthworm meal has been used for poultry, chicken and fish in diets because it can substitute for fishmeal in diets (Bay 2002). The studies had done in India (Kale et al., 1982) and Guerro, 1983) shown that earthworm have successfully converted animal manure into a good source of protein as supplementary in the diet for animal and the weight gain of Tilapia was higher when given a diet with a supplement of earthworm than when given a fish meal supplement. The study by Nguyen Duy Quynh Tram et al., (2007) on growing catfish fed a diet containing 30% CP from trash fish as control diet and with the CP from trash fish replace by earthworm of 25, 50, 75 and 100% showed that weight gain, daily weight gain, FCR and specific growth rate were highest for earthworm 75% at 41.3 g, 0.69g/day, 1.53 g, feed/g gain and 4.5%/day respectively and the lowest growth parameter were in treatment without earthworm. However, the study using earthworm for frog is little study.

Water spinach is available naturally in the rainy season or it can be planted in the rainy and dry seasons. The two types of water spinach; land and aquatic, are commonly cultivated by farmers. When water is not a constraint factor, aquatic water spinach has the capacity to produce foliage for longer period. Aquatic water spinach produces up to 40 ha^-1 cut^-1 when applied 200 kg of nitrogen ha^-1 year^-1 (CelAgrid, unpublished data). Beside high yield, water spinach have high protein 27.1% and have been use for feeding pig and growth rate were respond with level of water spinach in the diets (chhayty et al 2005ab,2006,2007).  

Other protein source is duckweed, it can growth well in waste water and protein are variable depend on fertilizer and rang from 35-40% in the dry matter (Leng et al 1995; Rodriguez and Preston 1996; Nguyen Duc Anh et al 1997; Le Ha Chau 1998). The balance of essential amino acids in duckweed is considered to be comparable to that in soy bean meal (Muztar et al 1976; Rusoff et al 1980). The protein in duckweed is more digestible than in cassava leaves (Nguyen Van Lai and Rodriguez 1998).  

The aims of the present study is using earthworm, water spinach and duckweed as protein sources for growing frogs with basal diet of rice bran and broken rice

Materials and methods

Location  

The experiment was carried out at the Center for Livestock and Agriculture Development (CelAgrid), located in Preah Theat village, Sankat Rolous, Khan Dangkor about 25km from Phnom Penh City, Cambodia during rainy season from 05^th July to 03^rd October 2011.

Ponds preparation

A total of 18 ponds were organized under tree and without roof with area of 2 m² (2m x 1m) and 0.4m deep in each pond. All ponds were lined with white plastic in order to avoid filtration of water. Small wood post each 1 m high was erect at the four corners of each pond and a plastic net were placed around the poles to make a fence for the pond. Electric light were connected at night on the surface of the pond (0.8m high) for light supplementation treatment, while without light supplementation, plastic net were used to cover the surface of the pond in order to avoid protein source (insect) at night.

Experimental frog

A total of 144 frogs were bought from a farmer’s farm in Kandal province. All frogs were adapted to experimental area and feed for 10-15 days before started experiment. Frog stocking densities were 4 frogs per m².

Experimental feed and feeding 

The California earthworm and duckweed were cultured at CelAgrid farm, while water spinach was purchased from farmers. Rice bran and broken rice were purchased from local rice mill around the center. Water spinach was chopped very small pices (about 0.5-1mm) before feeding to frogs, while duckweed was wilted overnight before feeding. Earthworm after the harvesting was chopped from 2-3cm and then mixing with other ingredients and very homogenized.

 The frogs were fed twice per day at 07.00 h and 18.00 h. All feed ingredients were mixed together before feeding. The quantities were fixed at 10% of the body weight. All experimental ingredients were mixed and very homogenized. The experiment was conducted in the raining season during 90 days of experiment.  

Experimental design 

The experiment design was Completely Randomized Design (CRD) with 3*2 factorial arrangements of 6 treatments and three replications within each treatment, the first factor was protein sources from earthworm (EW) alone, duckweed mix with earthworm (DW-EW) or water spinach mix with earthworm (WS-EW) and second factor was with or without light supplementation at night.  

Protein sources

• EW      :  Earthworm + broken rice and rice bran
• DW-EW: Duckweed + earthworm + broken rice and rice bran
• WS-EW:  Water spinach + earthworm + broken rice and rice bran

Light supplement at night

• With
• Without

Treatment combination

• Earthworm + broken rice + rice bran and electric light suppmentation
• Duckweed + earthworm + + broken rice + rice bran and electric light suppmentation
• Water spinach + earthworm + broken rice + rice bran and electric light suppmentation
• Earthworm + broken rice + rice bran and without light suppmentation
• Duckweed + earthworm + broken rice + rice bran and without light suppmentation
• Water spinach + earthworm + broken rice + rice bran and without light suppmentation

Measurements and chemical analysis

The amounts of feed offered were recorded daily. All frogs/pond (8 heads) were weighed every 10 days during the 90 days of the experiments. Individual daily weight gains were calculated by the regression of live weight on time in days. Feed conversion ratio was calculated from individual daily DM offer/intake and live weight gain. 

Samples of ingredients such as earthworm, duckweed, water spinach, rice bran and broken rice were taken for the analysis of nitrogen, using a Foss-Tecatur kjeldahl apparatus (AOAC 1990), and for dry matter by oven (Undersander et al 1993). 

The oxygen level of the pond water was measured every week, two times at 7 am and at 2 pm. “Winkler” test-kits (Aquamerck) was used for the measurement. The pH of the pond water was also measured every week, two times at 7 am and at 2 pm by glass electrode. Water temperature was measured every week, three times a day in the morning at 7 am, at 12 am and in the evening at 5 pm.

Statistical analysis 

The data from feed offer, growth rate, feed conversion, and yield and survival rate were analyzed by the General Linear Model in the ANOVA option of the software in Minitab 2007 (version 15.1.2). Sources of variation were protein source, light supplementation and interaction between protein source * light supplementation and error. When the F-test was significant at P<0.05, pair wise comparisons were performed using Turkey’s procedure (Minitab Statistical Software). The model used was:

Y_ij = μ + E_i + L_j + E_i*L_j + e_ijk

Where, Y = Dependent variable

μ = Overall mean

E_i = Protein sources effect

L_j = With or without light supplementation effect

E_i*L_j = Interaction between protein sources * light supplementation

e_ij = random error

Results and discussion

General

The experiment was conducted in rainy season and we could not collected feed refusal because most of feed refusals were flow to frog ponds. Most of feed refusals were broken rice, rice bran and water plant (water spinach or duckweed) but earth worm were consume quickly by frog and no refusal.   

Feed offer 

Total feed offer in fresh basis were high on the mixture DW-EW, then WS-EW and the lowest was EW treatment alone (P<0.001), while offer in DM basis were not different of DW-EW and EW but lowest was found in WS-EW treatment (P<0.001), meanwhile, crude protein offer was high for EW, then DW-EW and lowest was WS-EW (P<0.001). In term of light supplementation was higher than without light supplementation. The different in feed offer could be effect by different on live body weight during the periods of experiment. A recommendation by LARReC, (2001) reported that the lowland frog require similar nutrients as other aquatic animal species, especially those that are carnivorous, to maintain normal growth and metabolic function. The major nutrients such as protein, lipids, essential fatty acids, carbohydrates, vitamins and minerals are required for normal growth. A study by Thongyount (2004) reported that tadpoles from 1-30 days require protein at 38% in DM and reduce to 32% for small frogs (30-60 days) and 26% for fattening frog (60-90 days). Artificial feed of 35 % crude protein supported faster growth in Rana pipen tadpoles than young tilapia fish of 23 % protein (Ling et al., 2003). According to Martinez et al., (1993) mention that commercial feeds with 39% protein feed is suitable for frogs in intensive culture. Somsueb and Boonyaratpalin (2001) also reported that the optimum protein content of diets for raising frogs intensively is 37% in DM. Protein recommendations above are ranged in present study which balance of 30% in DM basic in three diets, however, it was lower than study of Latsamy Phounvisouk and Preston (2007) who use 53.5-55.1% in DM basic in the diets but the high value because of they used only earthworm alone or earthworm (90.2% in the diets DM basis) mixture with fresh duckweed (9.76% in the diets in DM basis).  

Weight gain and feed conversion ratio 

The live weight gain (LWG) were found the best in all period of experiments when the frogs fed EW diet rather than WS-EW or DW-EW treatment (P<0.05), by the ways, light supplementation at night also effect on LWG compared with without light supplementation treatment (P<0.05) but excepted from 30-60 days (P>0.05). Latsamy Phounvisouk and Preston (2007) study on fly larvae, earthworms and duckweed as feeds for frogs in an integrated farming system showed that daily LWG was 2.10g when the frogs fed only earthworm alone (100% in DM basis) but daily LWG was increased to 2.80g when the frogs fed earthworm (90.2% in the diet, DM basis) mixed with fresh duckweed (9.76% in the diets, DM basis). The report was contracted with present study which showed that LWG was high in earthworm (2.62g/day) treatment compared with earthworm mix with duckweed (1.91g/day) treatment and by the ways, earthworm that used in present study was lower level (20-40% in the diets, DM basis) and balance of crude protein in the diets (30% in DM) compared with Latsamy Phounvisouk and Preston (2007) used high amount of earthworm (90-100% in the diets, DM basis) and high crude protein in the diets (53.5-55.1%, DM basis). Other possible of contraction with daily weigh gain of study of Latsamy Phounvisouk and Preston (2007) could be escape of some earthworm to the pond, while the present study, earthworm were chopped and mixed with other ingredients. The present study showed growth rate much higher (2.62g/day) than study on catfish (0.69g/day) given diets containing rice bran (60%), trash fish (10%) and earthworms (30%) (Nguyen Duy Quynh Tram et al., 2007). Support for the high nutritive value of the earthworms for frogs is the finding in the study of Nguyen Duy Quynh Tram et al (2007) that the growth rates of the Catfish increased from 0.39 to 0.69 g/day when earthworms replaced 75% of the trash fish in the diet. Rodriquez-Serna et al., (1996) were reported that the growth rate of frogs fed pellet feed and house fly larvae in Mexico gain only to be 0.63 g/day which is less than recorded for the house fly larvae diets in study of Latsamy Phounvisouk and Preston (2007) (0.89 g/day for larvae alone and 1.14 g/day for larvae plus duckweed). 

In figure 1 (0-30days) & 2 (30-60days) showed that there were not interaction between protein sources and light supplementation (P>0.05) on daily LWG but in figure 3 (60-90 days) & 4 (0-90 days) showed that there were interaction in DW-EW and WS-EW treatment and supplementation with light at night but not for EW treatment in figure 3 or trend in figure 4.

Figure 5 showed that body weight at starting to 20 days were not much changed but started from 30 days toward, the daily weight gain were changed and highest for frog fed EW alone, then DW-EW but the lowest in WS-EW. In term of light also showed that no different at starting to 10 days but from 20 days toward the daily weight were better for frog supplement light at night compared without light supplementation (figure 6). 

Feed conversion ratio 

In overall, feed conversion ratio in DM basis was better for the mixture of DW-EW and EW alone and poorest was WS-EW treatment (P<0.001), while the light supplementation was not effect (P>0.05) but excepted period of 60-90 days, feed conversion ratio were better on light supplementation (P<0.05). The DM feed conversion ratio (FCR) in this study was higher than the study of Latsamy Phounvisouk and Preston (2007), the low FRC might be cause of the amount of feed offer were controlled according to the appetite of the frogs with the objective of avoiding feed residues, while in present study amount of feed offer were fix at 10% of body weight because the experiment was conducted in raining season and could not collect feed refusal. There were no interaction between protein sources and light supplementation on feed conversion ratio (P>0.05) but excepted from 60-90days which has interaction effect (P=0.037). 

Survival rate 

In table 6 showed that EW treatment has highest survival rate rather than mixture of DW-EW and WS-EW (P<0.05) but protein sources from forage (duckweed or water spinach) mix with earthworm were not significant different, meanwhile, light supplementation at night was effect on high survival rate compared with without light supplementation (P<0.005). However, survival rate in present study was lower than study of Latsamy Phounvisouk and Preston (2007) showed that the mortality of frogs was 2.48-3.26% only. High mortality or low survival rate in this present study might be some frogs escaped from ponds or ate by predators (snack) because dead frogs could not find in the pond in the whole experiment. There were no interaction between protein sources and light supplementation on survival rate (P>0.05). 

Frog yield 

In table 7 showed that total initial frog weight was not different (P>0.05), but after 90 days of experiments, total frog weight was increased (P<0.001) and net yield increase per pond and net yield increase per ha were found high in EW treatment rather than DW-EW and DW-EW (P<0.001), however, protein from forage mix with earthworm was not significant different. Meanwhile, light supplementation effect on high net yield compared with without light supplementation (P<0.001). The net increase in live weight in present study was ranged from 143.6-225.5g in 90 days and it was rang with study of Latsamy Phounvisouk and Preston (2007) (252 g in 90 days the frog eat earthworms plus duckweed) and Bounsong, (2001) (200g in 120 days that frog fed an artificial diet containing fish meal, dextrin, starch, rice bran, vegetable oil, tuna oil, vitamins, minerals, BHT and Choline chloride). There were no interaction between protein sources and light supplementation on frog yields (P>0.05). 

Water quality parameter  

The mean value of water temperature was 26, 30 and 28⁰C in morning, afternoon and evening respectively, this value rang with recommendation of Uodone (2004) that suitable temperature was 26.5 to 27.8⁰C. According to Diana et al., (1997) reported that water temperatures of 18^o-22^oC are generally suitable for rearing larval salamander species found in Laos, and temperatures of 18^o-22^oC are suitable for most species of frog. In term of pH value was range from 6.14 to 7.28 which similar recommendation from Uodone (2004) that ranged from 5.5-7.7. Dissolved oxygen was found high for EW treatment (P<0.05) but not different between DW-EW and WS-EW, meanwhile, DO in light supplementation was higher than without light supplementation (P<0.05), however, this value was ranged with study of Uodone (2004) (5.6-7.2).    

 Conclusions  

• Through the experiment conducted in 90 days can concluded that growth rates, survival and yield were best on treatment earthworm alone and light supplementation, while the mixture of duckweed with earthworm were improved but poorest was water spinach mix with earthworm. The net increase in live weight was 225.3 on the best diet (earthworms) was better than in one report concerning frogs fed an artificial diet (200 g in 120 days).

Acknowledgments 

The authors would like to express the gratitude to the MEKARN project financed by the SIDA-SAREC Agency. Student from Preak Leap agriculture colleague for helping and take care the experiment and Center for Livestock and Agriculture Development for providing resources for conducting this experiment.

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