J. Env. Bio-Sci., 2014: Vol.

28 (2): 293-298 ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

EFFECT OF ENRICHED MOINA AND MICROPARTICULATE DIET ON FATTY ACID

COMPOSITION OF PENGBA (OSTEOBRAMA BELANGERI) LARVAE
R. Ramesh1#, Kiran Dube1, A. K. Reddy1, P. V. Rangacharyulu2 and G. Venkateshwarlu1
Central Institute of Fisheries Education, Panch Marg, Off Yari Road, Versova, Andheri (West), Mumbai - 400 061,
Maharashtra, India1
Regional Research Centre, CIFA, Fish seed farm, Penamaluru, Poranki P.O., Vijayawada - 521 137, Andhra Pradesh, India2
email: rathodcifa@gmail.com#

Received: 25-09-2014 Accepted: 29-10-2014

In order to evaluate the effect of enriched Moina and microparticulate diet with different combinations on the tissue fatty acid
composition pengba larvae was studied. The feeding trial was conducted for the larvae using seven experimental diets consist of
Moina (enriched with algae and cod liver oil) and combination of enriched Moina and micro particulate diet. A total of thirty one fatty
acids were identified by GC-MS encompassing saturated, mono unsaturated, n-6 and n-3 poly unsaturated fatty acids. The n-6 and
n-3 PUFA showed a dramatic proportional increase in larval tissue relative to its proportional composition in both the live and
formulated diets. Dietary levels of the n-6 and n-3 fatty acids were similar among diets-4, 5, 6 (enriched Moina with cod liver oil and
algae). However, these fatty acids levels in the larvae were significantly higher in larvae fed combination of enriched Moina and
microparticulate diet (Diet-5 and Diet-6) compared to larvae fed other diets. Proportional increases of AA, EPA and DHA (20:4n-6,
20:5n-3 and 22:6n-3 respectively) in larvae relative to dietary HUFA, suggest important physiological roles for these fatty acids. The
increase in n-3/n-6 ratio vice versa demonstrated that larvae may require the higher EPA and DHA in their first feeding. The effect
of different feeding regimes of respective diets fed to pengba larvae based on fatty acid composition were clearly established by
principal component analysis.

Live feed has become the most important secondary producers
in aquatic food webs, and has been extensively used as high
quality natural food in hatcheries for the maintenance or
propagation of early larval stages of fishes for the past few
decades1. Moina has recently become a potential live feed,
with respect to its availability, in most of the natural water
resources and has a high nutritional value. This freshwater
zooplankton is classified as a cladoceran and it has been
documented as a potential live feed to substitute the use of
the more expensive Artemia and other seasonal zooplankton
such as copepods, in larviculture2.
In many hatcheries, common enrichment practices to enrich
fatty acids composition in Artemia and other live feeds such
as rotifers consisted of microalgae, microalgae paste, and
various marine fish oils3. Das et al. found that the nutritional
value of Moina could be similarly enhanced by enrichment
with various culture media as demonstrated in Artemia nauplii
and rotifer2. However, there is limited information, and no notable
study has been found on M. micrura enrichment, especially
on the aspects of culture media selection such as micro algal
enrichment concentration and appropriate treatment time.
Pengba, Osteobrama belangeri is a medium carp regarded as
the State Fish of Manipur, Northeastern states of India. The
fish can be considered as potential fish species for
diversification of carp culture due to its better taste and meat
quality. As part of studies on development of breeding and
production technology of pengba, the evaluation of nutritional
requirement of this species needs to be determined. So far no
attempts were made to improve the fatty acid composition of
pengba larvae with respect to live feed enrichment and artificial
diets for their better growth and survival. In this background,
the study was conducted to emphasize the effect of co-feeding
on fatty acid composition of larval pengba with Moina and
microparticulate diet.
MATERIALS AND METHODS
Moina culture and enrichment
Mass culture of Moina and enrichment was followed the method
of Parakarma et al4. The composition of prepared emulsion for
enrichment of Moina is given in Table -1.
Experimental fish and feeding strategies:Pengba larvae
(three days old post yolk sac) were obtained from hatchery
upon successful breeding of pengba brooders. Total 2,100 one
week old larvae (average weight 0.15±0.01 mg) were randomly
 Effect of Enriched Moina and Microparticulate Diet on Fatty Acid (294)

[(Diet-1 (Moina%), Diet-2 (Moina + MD), Diet-3 (Moina enriched with cod liver oil), Diet-4 (Moina enriched with cod liver oil + MD),
Diet-5 (Moina enriched with algae), Diet-6 (Moina enriched with algae + MD) and Diet-7 (Microparticulate diet)].

Fig. 1. Bi-plot of distribution in fatty composition of larvae of pengba (Osteobrama belangeri)

fed with different feeding regimes

Table-1. Composition of emulsion media used for Moina enrichment

distributed into 21 smaller rectangular tanks (60cm height,
74cm length, 44cm width) at the density of 100 larvae per
tank. The feeding trial was conducted for 35 days with three
replicates of each treatment following a completely
randomized design. Fish larvae was fed with seven
experimental diets namely Moina (Diet-1), Moina + MD (Diet-
2), Moina enriched with cod liver oil (Diet-3), Moina enriched
with cod liver oil + MD (Diet-4), Moina enriched with algae
(Diet-5), Moina enriched with algae + MD (Diet-6) and
Microparticulate diet (MD) (Diet-7). The larvae were fed ad
libitum daily twice at 08:00 and 17:00 h. Uneaten feed was
siphoned daily to maintain optimum water quality. The water
temperature was recorded 22.2±2.8 °C and water quality
parameters such as Dissolved oxygen level was 6.2±0.66 mg/l,
pH 7.2±0.50, alkalinity 152.5±4.56 mg/l, total hardness 300-
400 mg/l and NH4 0.1 mg/l were recorded throughout the
experiment.
Total lipid and Fatty acid ethyl esters (FAME)
Total lipids of enriched and micro particulate diets were extracted
(295) Ramesh, Dube, Reddy, Rangacharyulu and Venkateshwarlu

Table-2. Fatty acid composition (% of total fatty acids) of different diets fed to pengba larvae

[ND: Not Detected; CLO:Cod liver oil; MD: Microparticulate diet]

 Effect of Enriched Moina and Microparticulate Diet on Fatty Acid (296)

Table-3. Fatty acid composition (% of total fatty acids) of pengba larvae fed with different
feeding regimes

[ND: Not Detected]

(297) Ramesh, Dube, Reddy, Rangacharyulu and Venkateshwarlu
following the method of Folch et al5. The AOAC method was
followed to esterify the lipid extract6. Fatty acid methyl esters
(FAME) were analyzed using a Shimadzu QP2010 Quadruple
Gas Chromatography Mass Spectrometer (GCMS) equipped
with a Carbowax (30 m x 0.25 mm ID; 0.25- μ m film thickness)
capillary column (Cromlab S.A.). Helium was used as the carrier
gas. The values of fatty acids are present in peak area
percentages of total identified fatty acids. Statistical
analysis.The multivariate data on fatty acid composition of
larvae subjected to different feeding regimes with enriched
Moina and microparticulate diets were analyzed by principal
component analysis (PCA) using software Unscrambler
(Version 9.5, CAMO, Norway).
RESULTS AND DISCUSSION
Fatty acid composition of diets: The fatty acid composition
of Moina (enriched with cod liver oil and micro algae) and
microparticulate diet is summarized in Table-2. From the fatty
acid data, total saturated fatty acids (SAFA), total
monounsaturated fatty acids (MUFA), sum of n-6 and n-3 poly
in saturated fatty acids (PUFA) and the ratios of n-3/n-6, n-6/
n-3 were calculated. The SAFA level was highest in unenriched
Moina (46.49%) and lowest (32.05%) was found in
microparticulate diet. MUFA level was highest (38.8%) in Moina
enriched with cod liver oil, whereas lowest (30.87%) was
observed in unenriched Moina. However, the highest n-6 and
n-3 PUFA were found in microparticulate diet (22.64%) and
enriched Moina (11.07%, CLO and 11.69%, algae) respectively.
Fatty acid composition of larvae:The fatty acid profiles of
larvae fed with different dietary feeding regimes were presented
in Table-3. In pengba larvae, the SAFA level was highest in
Diet-1 (57%) and lowest (46.23%) was found in Diet-6. MUFA
level was highest (38.8%) in Diet-2 and lowest (30.87%) was
observed in Diet-7. The highest n-6 and n-3 PUFA were found
in Diet-6 (10.22%) and Diet-7 (8.47%) respectively. The most
common SAFA was dominated by palmitic acid (29.35-39.12%)
and stearic acid (4.12-6.21%). Among MUFA, oleic acid (18:1n-
9) was the most abundant comprising of 18.13-33.27% of the
total fatty acids followed by palmitoleic acid (16:1n-7) which
formed 0.54-4.24%. Linoleic acid (18:2n-6) and Arachidionic
acid (20:4n-6) were the principal n-6 PUFA at a level of 1.04-
6.46% and 0.19-3.24% of total fatty acids respectively. Linolenic
acid (18:3n-3), Eicosapentaenoic acid (EPA, 20:5n-3) and
Docosahexaenoic acid (DHA, 22:6n-3) were the major n-3
PUFA identified, accounting 0.32-3.66%, 0.34-2.68% and 0.32-
1.36%, respectively.
In the present study, the fatty acid composition of fish larvae
is directly influenced by the different dietary treatments. The
fatty acid composition of fish in general is known to be
influenced by the fatty acid composition of dietary lipid7. Further,
tissue fatty acid composition is affected by size or age of
animals8, food deprivation9 and non-dietary factors including
environmental temperature10. The presence of high amount 16:0
and 18:1n-9 fatty acids in the present study supported by
Sargent et al, who proposed that 16:0 and 18:1 n-9 are the
most preferred fatty acids for the mitochondrial β -oxidation
and thus the source of metabolic energy in fish11. The results
are in agreement with Faulk and Holt found that enriching
rotifers and Artemia with live algae can enhance the HUFAs
content in the live feed and consequently improve the growth
and survival of cobia larvae12.
PUFAs in live organisms like microalgae are much more stable
than in commercially available emulsion formulations. It is
significant that the levels of AA, EPA and DHA found in enriched
live microalgal cells more closely resemble larval natural diets.
In particular, the competitive interactions between AA and EPA
are important in the formation of eicosanoids13. The present
study the AA levels are very less in the body tissues which
contrast to earlier studies conducted by Copeman et al in
yellowtail flounder where AA levels were increased in body
tissues in spite of lower AA level in the diet14. The ratio of n-6
and n-3 PUFA were ranged from 0.27 to 3.66 revealed that the
importance of EPA and DHA concentrations, were influenced
in the dietary treatments. High ratio of n-6 and n-3 PUFA were
found in larval tissues suggested that EPA and DHA contributed
in their biological activities. These findings were supported by
earlier workers Watanabe et al. and Koven et al. proposed
notably in marine fish species 15, 16.
To gain better insight into the data structure, principal
component analysis (PCA) was performed on the fatty acid
composition of larval samples obtained at seven different
feeding regimes. The projection of variables and different diets
onto the first two principal components was given in Fig. 1 in
the form of bi-plot. The analysis explored in a two-principal
component (PC1 and PC2) model that described 71% of the
total data variability. The pengba larvae fed with different diets
were separated based on fatty acid composition and
demonstrated that fatty acid profile of pengba larvae were clearly
 Effect of Enriched Moina and Microparticulate Diet on Fatty Acid
influenced by different feeding regimes. PC1, which explained 4.
44% of total information in the data, has clearly separated
diet-5 and diet-6 from other diets. Further, it can be clearly 5.
noticed from the bi-plot, that the pengba larvae fed with diet-5
and diet-6 were rich in n-6 and n-3 fatty acids such as 18:2n- 6.
6, 20:2n-6, EPA, DPA and DHA, were found to be responsible
for causing the differences among the samples. The present
study was supported by Singh et al., where the PCA showed
7.
the differences in the fatty acid profiles of fish fry fed with
different dietary groups enriched with live feed and formulated
8.
diets and was found to be responsible for causing the
differences among the samples17.
In conclusion, enrichment of Moina (algae and cod liver oil)
9.
combination diets contained relative proportional of fatty acids
along with their favorable n-3/n-6 ratios which are directly
10.
proportional to the larval tissue fatty acids. The EPA and DHA
found in pengba larvae fed with various dietary treatments
assume that they played a significant role in their physiological 11.
activity. The co-feeding method was effective in improving the
nutritional values of larvae for their better growth and survival. 12.
13.
REFERENCES
1. Cary, T.L., Chandler, G.T., Volz, D.C., Walse, S.S. and Ferry, 14.
J.L. (2004). Environ. Sci. Techn., 38:522.
2. Das, S.K., Tiwari, V.K., Venkateshwarlu, G., Reddy, A.K., 15.
Parhi, J., Sharma, P. and Chettri, J.K. (2007). Aquaculture, 16.
269:464.
3. Papandroulakis, N., Divanach, P. and Kentouri, M. (2002). 17.
Aquaculture, 204, 45.
(298)
Parakarma, M.G.I.S., Rawat, K.D., Venkateshwarlu, G. and
Reddy, A.K. (2009). J. Ind. Fish. Assoc., 36:9.
Folch, J.L., Sloane, M. and Stanley, G.H.S. (1957). J. Biol.
Chem., 226: 497.
AOAC (1995) In: Official Methods of Analysis of the
Association of Official Analytical Chemists, AOAC,
Arlington, VA.
Torstensen, B.E., Lie, O. and Froyland, L. (2000). Lipids,
35:653.
Kiessling, A., Pickova, J., Johansson, L., Asgard, T.,
Storebakken, T. and Kiessling, K. (2001). Food Chem.,
73:271.
De Silva, S.S., Gunasekera, R.M., Collins, R., Ingram,
B.A. and Austin, C.M. (1997). J. Fish Biol., 50 (5): 992.
Bell, M.V., Henderson, R. J. and Sargent, J. R. (1986).
Com. Biochem. Physiol., 83B: 711.
Sargent, J.R., Tocher, D.R. and Bell, J.G. (2002). In: Fish
nutrition, Academic Press, San Diego, CA, pp. 181.
Faulk, C.K., Holt, G.J. (2005). Aquaculture, 249: 231.
Harel, M. and Place, A.R. (2003). Comp. Biochem.
Physiol., 135B: 83.
Copeman, L.A., Parrish, C.C., Brown, J.A. and Harel, M.
(2002). Aquaculture, 210: 285.
Koven, W. (2003). Israel J. Aquacult., 4: 283.
Watanabe, T., Kitajima, C. and Fujita, S. (1983).
Aquaculture, 34:115.
Singh, S.K., Mishra, U., Roy, S.D., Chadha, N.K. and
Venkateshwarlu, G. (2012). J. Aquacult. Res. Dev., 3:143.