IJERD – International Journal of Environmental and Rural Development (2015) 6-1

Research article
erd

Growth Performance and Feed Utilization of Common
Lowland Frog (Rana rugulosa Wiegmann) Fed with
Supplementation by Bromelain Extracted from Pineapple
Feed
RUNGKAN KLAHAN*
Faculty of Agricultural Technology, Phetchaburi Rajabhat University, Phetchaburi, Thailand
Email: rukiirun@windowslive.com

BANYAT SIRITHANAWONG
Faculty of Agricultural Technology, Phetchaburi Rajabhat University, Phetchaburi, Thailand
Received 15 December 2014

Accepted 7 April 2015(*Corresponding Author)

Abstract This study aimed to determine the effect of bromelain extracted from the crownof pineapple supplemented in diet on growth performance and feed utilization of common
lowland frog. Diets were isonitrogenous at 35% protein and 3,000 KcalKg-1 supplemented
at 0, 0.25, 0.5 and 1 mL bromelain extraction g-1 of feed. Frog with an initial weight of 0.25
± 0.1 g were fed on feed trial in triplicate groups for 120 days. The results showed that theenzyme extracts did not effect growth performance but enhancement the survival rate withall levels (P<0.05) especially at 1 mL/g feed had the highest feed utilization and protein
efficiency ratio (PER). The percentage of edible flesh of 0, 0.5 and 1 mL/g feed group werehigher than the 0.25 mL/g feed group. These results demonstrated that bromelain extracted
supplementation of all levels improved survival rate and feed utilization and PER especiallyat 1 mL/g feed.
Keywords common lowland frog, bromelain, pineapple, growth performance, feedutilization

INTRODUCTION
In 2011 the total area of pineapple cultivation in Thailand is around 104,571 hectare (646,000 rai).
Prachuapkhirikhan has the largest share at 47% of the total pineapple cultivated areas of thecountry; other major growing provinces are Chonburi, Rayong, Trat, Chacheongsao, Kanchanaburi,Phetchaburi and Ratchaburi. Thailand is the world’s number one exporter of canned pineapple. In
2011, Thailand exported canned pineapple totaling 641,167 tons (or 36,430 FCLs; 1 FCL = 17.6tons), with the total value of 669.4 million dollars. Thailand’s main export markets are UnitedStates, EU and Russia. The waste from pineapple (peel, core, stem and crown) generally is aboutfor 50% of the whole fruit that causing a serious environmental problem (Ketnawa et al., 2012).
The utilization of waste as feed additive in aquatic animal feed is the alternative way for handlingthe great deal of waste problem.
Bromelain is a mixture of proteolytic enzyme, belong to a group of protein digesting enzyme
derived from the fruit or stem of pineapple (Ananas comosus L.) (Bhattacharyya, 2008; Bala et al.,2012). Bromelain is the most effectiveness in reduction of inflammatory and decreasing swelling asa natural anti – inflammatory enzyme. It’s a mixture of different thiol endopeptidase and othercomponents like phosphatase, peroxidases, glucosidases, glycoproteins, cellulose, carbohydrataseand several protease inhibitors (Bhattacharyya, 2008). Bromelain is not very specific in action but
preferentially cleaves glycol, anayl and leucyl bonds. The bromelain actions on protein
predigestion, diarrhea, digestive aid, anti thrombotic, oedema treatment and osteoarthritis andpromotes the absorption of antibiotic drug. (Corzo et al., 2012). It is contains, among othercomponents, various closely related proteinases, anti - edematous, anti - inflammatory,Ⓒ ISERD58

IJERD – International Journal of Environmental and Rural Development (2015) 6-1

antithrombotic and fibrinolytic activities. Moreover, due to its safety and lack of undesired sideeffects, bromelain has earned growing acceptance and compliance among patients as aphytotherapeutical drug. The pharmacological properties depend on the proteolytic activity only.
Bromelain contains several distinct cysteine proteinases that have similar but distinct amino acidsequences, as well as differences in proteolytic specificity and sensitivity to inactivation (Hale et al.,2005). From the mentioned properties, the bromelain from pineapple should be used as feed
additive in frog feed. Because of the gastrointestinal tract of tadpole and juvenile frog are similar tofish larvae that required feed all the time that causes digestive aid and dead in final. In addition, atthe disappearence of the tail stage the frog does not eat feed experiences mortality also. This
problem is often found in aquatic animal larvae. The digestion depend on digestive enzyme such asprotease, amylase and lipase especially protease because the frog is carnivorous and has the highprotein requirement (Duellman and Trueb, 1994). From the properties of bromelain and theproblem on digestion of frog, bromelain should be used as feed additive in frog feed for improving
the digestion and depressing the digestive aid and the mortality.
OBJECTIVE
The purpose of this study was to investigated the ability of diet supplemented with bromelian
extraction from pineapple on feed utilization, growth performance, percentage of edible flesh andsurvival rate of common lowland frog.
METHODOLOGY
Experimental Frogs
Frogs at 10 days old (initial mean weight 0.25 ± 0.10 g), produced in Petchaburi Rajabhat
University aquaculture program were randomly stocked into 1 m2 cement tank at a density of 30
frog per tank. Frogs were acclimated for 1 week with control feed (pellet feed without bromelainsupplementation) before start the experiment.
Raw Material and Crude Extract Preparation
The pineapple (Ananus comosus L.) (“Batavia”) was collected from plantation in the Ratchaburiprovince, Thailand. The fruit was washed and air dried. The crown (Fig. 1) was separated andchopped to small pieces. This study used the crown because from the previous study it was foundthat the bromelain activity (from waste: peel and crown) was highest in crown. Small pieces ofcrown were blended with cold distilled water at a 1:1 ratio for 5 min and the blended liquid wasfiltered through a sieve (250 µm) and then centrifuged at 10,000 x g at 4 oC for 20 minnutes(Ketnawa et al., 2012). The supernatant (crude extract) will be collected to mix with feed.
Experimental Diets
Frogs were fed with floating pellet diet (35% protein, 3000 Kcalkg-1 energy). Proximate
composition of the experimental diets is determined by analysis (Kjeldahl method for crude protein,Soxhlet method for lipid, Detergent method for crude fiber, Oven drying method for moisture anddetermination of ash by muffle furnace) (AOAC, 2000). Bromelain extraction was used at 0, 0.25,
0.5 and 1 mg-1 diet as a feed supplement. In the preparation of experimental diet, bromelain weremixed with a diet and control diet was also mixed with 1 mL water for 1 g feed. These pelleted
diets were coated with fish oil at 1% and then incubated at 37 oC for 1 hour then dried in the hot air
oven at 90 oC for 2 hours. The dry pellets were placed in covered plastic bag and stored at roomtemperature.

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ABFig. 1 Pineapple whole fruit (A) and crown proportion (B)Experimental Procedure
Each of the four experimental diets was randomly assigned to triplicate groups of frog and all thegroups were fed with the prepared diet at 5% body weight twice daily for 120 days. Also, water
was changed for every two days for 100% throughout the study.
Analytical MethodDuring experiment, the mortality was recorded daily and frog in each tank were monthly countedand weighed individually. Growth rate were monitored to determine the final weight, weighed gain,
Specific Growth Rate (SGR), average daily gain, survival rate, feed intake, Feed Conversion Ratio(FCR) and Protein Efficiency Ratio (PER) were calculated according to Castell and Tiews (1980).
At the start of experiment, 50 frogs randomly were dried for the determination of body proximate
composition. At the end of the feeding trial, 10 frogs from each group (n = 30 frogs/group) wereanalyzed for final whole body proximate composition. Proximate composition of body wasanalyzed following the AOAC (2000) method.
Statistical Analysis
In the experiment, all data were analyzed by one-way analysis of variance (ANOVA) followed by
Duncan's multiple range tests. A significance level of P<0.05 was used.
RESULTS AND DISCUSSION
Growth Performance
The result of the growth trial showed that all groups were not significantly different (P>0.05)(Table 1). The mean final weight, weigh gain, ADG and SGR of different diets ranged between
84.94 – 98.62 g/f, 84.60 – 98.37 g/f, 0.91 – 1.11 g/f/day and 2.54 – 2.79 %, respectively. In thecontrary, survival rate in all groups fed with supplemented bromelain feed showed significantly thehighest survival rate (P<0.05). Bromelain is contains, among other components, various closely
related anti - edematous, anti - inflammatory, antithrombotic and fibrinolytic activities that promote
the great health effected on the high survival rate. The growth rate was similar with among group
because frog is carnivorous which the density effected on the growth rate. The amount of frog after
fed with supplement bromelain feed for 120 days has the high density proportion when comparedwith no supplement bromelain feed group so make the growth rate not outstanding.

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Table1 Growth performance of frog fed with supplementation diet by bromelain extracted at120 days
Growth performance
Initial weight (g/f)
Final weight (g/f)
Weight gain (g/f)
Average daily gain (g/f/day)Specific growth rate (%/day)Survival rate ( % )a,b,c00.24 ± 0.02
98.62 ± 3.71
98.37 ± 3.73
1.11 ± 0.13
2.79 ± 0.19
75.00 ± 2.36b

Bromelain supplementation (mL/g feed)
0.25
0.5
1
0.25 ± 0.01
0.25 ± 0.01
0.26 ± 0.00
84.94 ± 2.55
89.92 ± 5.76
87.23 ± 6.75
84.70 ± 2.54
89.66 ± 5.76
86.79 ± 6.74
1.11 ± 0.39
1.09 ± 0.25
0.91 ± 0.07
2.74 ± 0.03
2.72 ± 0.28
2.54 ± 0.08
85.55 ± 5.09a
92.22 ± 1.92a
91.11 ± 7.69a

Means within a row with common superscript are significantly different (P<0.05). n = 30

Feed Utilization
The result of feed utilization of frog fed with supplementation with bromelain at different level for
120 days showed that diet containing 0.25 – 1 mL of bromelain per 1 g feed were significantly
higher (P<0.05) than controlled group especially at 1 mL of bromelain per 1 g feed group resulted
in the highest feed utilization i.e. the best FCR, FCE and PER and the worst feed intake (Table 2).
Bromelain is a mixture of proteolytic enzymes, belonging to a group of protein digesting enzyme(Bhattacharyya, 2008 and Bala et al., 2012). It’s a mixture of different thiol endopeptidase andother components like phosphatase, peroxidases, glucosidases, glycoproteins, cellulose,
carbohydratase and several protease inhibitors (Bhattacharyya, 2008 Maurer, 2001). On the other
hand, the highest caseinolytic activity at a pH 5.5 - 8 (Ketnawa et al., 2012; Corzo et al., 2012 andBala et al., 2012) these cause make it has high digestibility efficiency. In addition, bromelain has
been successfully used as a digestive enzyme following pancreatectomy, in case of exocrine
pancreas insufficiency and the advantage is it has activity in the stomach as well as the small
intestine which also shown to be an adequate replacement of pepsin and trypsin in case ofdeficiency (Bhattacharyya, 2008).
Table 2 Feed utilization of frog fed with supplementation diet by bromelain extracted at 120days
Feed utilization
Feed intake (g/f/day)Feed conversion ratio
Feed conversion efficiency (%)
Protein efficiency ratio
a,b,c

0
1.56 ± 0.02a
1.64 ± 0.52a
74.43± 8.03b
1.84 ± 0.51b

Bromelain supplementation (mL/g feed)
0.25
0.5
11.05± 0.15bc
1.17 ± 0.24ab
0.65 ± 0.00c
1.18± 0.14b
1.47 ± 0.43ab
0.80 ± 0.09cbb
85.42± 9.81
81.59±12.01
133.99±0.21abb
1.73± 1.23
2.04 ± 0.55
3.60 ± 0.38a

Means within a row with common superscript are significantly different (P<0.05). n = 30Carcass Composition
The effect of different bromelain supplementation level on carcass composition showed in Table 3.
The results showed that there was no significant difference on percentage of bone and skin, visceraand fat and hepatosomatic index (HSI) (P>0.05) but it effected on percentage of edible flesh(P<0.05) that showed the highest on control group, 0.5 and 1 ml/g feed and has the lowest in 0.25
mL/g feed group. The composition of the amino acid (AA) mixture that was significantly influence
the composition of the fish free amino acid (FAA) pool. However, when fish are fed a feed
supplemented bromelain, a faster absorption of FAA may lead to transient AA imbalances andconsequently to decreased protein utilization if crystalline AA are used to supplement dietaryprotein.

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Table 3 Carcass composition of frog fed with supplementation diet by bromelain extracted at
120 days
Carcass composition (%)
Edible flesh
Bone and skin
Viscera and fat
Hepatosomatic index
a,b,c031.58 ± 1.34a
41.71 ± 0.37
28.84 ± 2.55
6.04 ± 0.12

Bromelain supplementation (mL/g feed)
0.25
0.5
127.63 ± 1.69b
28.72 ± 0.54ab 30.21 ± 0.48ab
43.36 ± 1.46
44.30 ± 1.97
41.10 ± 2.07
29.01 ± 1.81
28.77 ± 2.05
31.49 ± 0.14
6.38 ± 0.10
5.59 ± 1.06
6.01 ± 0.82

Means within a row with common superscript are significantly different (P<0.05). n = 30

CONCLUSION
Bromelain extracted from pineapple could be used as frog’s survival rate promoter. Thesupplementation bromelain all levels improved survival rate, feed utilization and PER especially at
1 mL/g feed. It can be concluded that bromelain extracted from pineapple’s crown is useful for
aquaculture and environment to get rid of the waste from agriculture.
ACKNOWLEDGEMENTS
This research was supported by the grant of Phetchaburi Rajabhat University.
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Castell, J.D. and Tiews, K. 1980. Report on the EIFAC, IUNS and ICES. Working group on thestandardization of methodology in fish nutrition research, Hamburg, Federal Republic of Germany. 2123 March, EIFAC Technical Paper, 26 p.
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