Effects of Vegetable Oil Source and Dietary Vegetable-fish Oil Ratio on the Histological Alterations of Liver and Intestine of Juvenile Malaysian Mahseer, Tor tambroides
Mohd Salleh Kamarudin
For many years, fish oil has been used as the major source of lipid in aquafeed production. However, the replacement of this marine origin ingredient with a sustainable resource such as vegetable oils is critically important. On the other hand, most of the vegetable oils are high in omega-6 polyunsaturated fatty acids while a diet rich in these fatty acids can increase liver lipid deposition in some fish. This study was conducted to investigate the effects of partial or total replacement of fish oil with different vegetable oil sources (sunflower and palm) on the histological alterations in the liver and intestine of juvenile Malaysian mahseer, T. tambroides. Five isonitrogenous and isocaloric diets were formulated in order to either completely or partially (50%) substitute fish oil with sunflower oil or palm oil. A diet without substitution was also used as a control. The effects of these diets on the histological alterations of liver and intestine of fish after a nine week feeding period were investigated. The results showed few inflammatory cells and lipid vacuoles in the liver of fish fed all the vegetable oil diets indicating minor hepatic steatosis. Lipid vacuoles were also observed in the intestinal wall of fish fed all the vegetable oil diets.
to cite this article:
Ehsan Ramezani-Fard and Mohd Salleh Kamarudin, 2013. Effects of Vegetable Oil Source and Dietary Vegetable-fish Oil Ratio on the Histological Alterations of Liver and Intestine of Juvenile Malaysian Mahseer, Tor tambroides. Asian Journal of Animal and Veterinary Advances, 8: 309-316.
July 02, 2012; Accepted: July 24, 2012;
Published: February 11, 2013
The mahseers (Tor spp.) are one of the large cyprinids occurred in Southeast
Asia (Bhatt et al., 2000). These fish are distributed
in many rivers, streams and lakes of mild hills of the Himalayan belt from Afghanistan
to Indo-China and Myanmar and form a significant stock of indigenous fish in
India, Nepal, Bangladesh and Pakistan (Bista et al.,
2002; Islam, 2002). Tor tambroides and Tor
douronensis, are two species of mahseers living in most major river systems
of Malaysia, in particular Perak, Terengganu, Pahang and Kelantan states. These
two sought-after species have a cultural and economic importance and are considered
as a high-valued fish in Sarawak, Malaysia (Ingram et
al., 2005). Similar to other mahseers in many countries, the natural
stock of this species is rapidly declining in recent years (Ingram
et al., 2007; Ramezani-Fard et al., 2011a)
and the government plans to develop its culture. Formulating a diet which meets
the nutritional requirements of Malaysian mahseer is an important factor for
launching the commercial aquaculture of this fish.
For many years, fish oil has been used as the major source of oil in aquafeed
production. Although fish oil is a rich source of n-3 long chain polyunsaturated
fatty acids (LC-PUFA) and prevents Essential Fatty Acid (EFA) deficiency in
farmed fish, the dependence of aquaculture on fishery-derived products is a
vicious circle and may not be sustainable in the future. Therefore, the replacement
of marine origin resources such as fishmeal and fish oil is at the core of a
heated global debate (Miller et al., 2007) and
the only practical substitute for fish oil is still vegetable oils. Most of
vegetable oils used in aquafeed industry are high in C18 Polyunsaturated Fatty
Acids (PUFA), particularly n-6 PUFA and low in LC-PUFA. A diet rich in n-6 PUFA
can increase liver lipid deposition in some fish (Robaina
et al., 1998). In these cases, histopathology provides information
to detect effects of irritants in various organs (Chezhian
et al., 2012). Development of large lipid vacuoles in the liver leads
to hepatocytes disruption and pyknotic nuclei occurrence. The current study
was carried out to investigate the effects of partial or total replacement of
fish oil with different vegetable oil sources (sunflower and palm) on the histological
alterations in the liver and intestine of juvenile Malaysian mahseer, T.
MATERIALS AND METHODS
Diet preparation: Five isonitrogenous and isocaloric diets with a lipid content of 5% were formulated in order to either completely or partially (50%) substitute fish oil with sunflower oil (diet S100 and S50) or palm oil (diet P100 and P50). A diet without substitution (containing 100% cod liver oil; CLO) was also used as a control. The compositions of the diets are shown in Table 1.
|| Ingredients and proximate composition of the experimental
|aDefatted Malaysian fishmeal (690 g kg-1
crude protein), bSeven Seas, c Sigma-Aldrich, dRefined,
bleached and deodorized palm olein, eVitamin premix (g kg-1
premix): Ascorbic acid: 45, Myo-inositol: 5, Choline chloride: 75, Niacin:
4.5, Riboflavin: 1, Pyridoxine: 1, Thiamin mononitrate: 0.9, Ca-pantothenate:
3, Retinyl acetate: 0.6, Cholecalciferol: 0.08, Vitamin K menadione: 1.7,
α-tocopherol acetate (500 IU g-1): 8, Biotin: 0.02, Folic
acid: 0.1, Vitamin B12: 0.001, Cellulose: 845.1, fMineral
premix (g kg-1 premix): KCl: 90, KI: 0.04, CaHPO4.2H2O:
500, NaCl: 40, CuSO4.5H2O: 3, ZnSO4.7H2O:
4, CoSO:, 0.02, FeSO4.7H2O: 20, MnSO4.H2O:
3, CaCo3: 215, MgOH: 124, Na2SeO3: 0.03,
NaF: 1, gCarbohydrates = Dry matter - [protein + lipid + ash]
Prior to mixing of the basal ingredients, fishmeal was defatted by soaking
in a mixture of chloroform and methanol (2:1, v/v) for 24 h in order to eliminate
any effect of residual LC-PUFAs originated from fishmeal (Kim
and Lee, 2004). Defatted fishmeal was then oven dried and mixed with the
other dry ingredients according to method explained by Kamarudin
et al. (2011).
Rearing and sampling: Fish obtaining and rearing methods for this experiment
are similar to those explained by Kamarudin et al.
(2011). Briefly, wild-caught T. tambroides juveniles of an initial
weight of 5.0±0.4 g (Mean±SD) were randomly distributed into 21
rectangular-shaped glass aquaria with the stocking density of 10 fish per aquarium.
Each aquarium was supplied with 65 L dechlorinated public utility water and
equipped with a recirculating system. The experiment was conducted for 9 weeks
and fish were fed twice per day close to visual satiety. At the end of experiment,
three fish per aquarium were sacrificed and dissected and their visceral organs
including liver were extracted. The extracted tissues were fixed in Bouins
solution at room temperature for 24 h, then washed and stored in 70% ethanol
until the wax embedding (Ramezani-Fard et al., 2011a).
Serial 5 μm sections were prepared, stained with haematoxylin-eosin and
slides were examined under a light microscopy (Zeiss Primo Star) fitted with
a digital camera (Canon A640).
Biochemical analysis: Analytical methods for the determination of crude
protein, crude lipid and ash contents as well as gross energy of experimental
diets have been already explained (Kamarudin et al.,
2011). Lipid of the experimental diets were extracted with a chloroform:methanol
(2:1, v:v) mixture (Folch et al., 1957; Ramezani-Fard
et al., 2011b) and transesterified with methanolic boron trifluoride
according to Kamarudin et al. (2011). Fatty acid
methyl esters were then separated on a fused silica capillary column (Supelco
SP-2330: 30 mx0.25 mm, film thickness 0.20 μm) in a gas chromatograph (Agilent
7890N) according to the method described by Ramezani-Fard
et al. (2012).
Fatty acid composition of all the experimental diets is shown in Table 2. Substitution of fish oil with sunflower or palm oil decreased total n-3 PUFA content of experimental diets. The highest content of 20:5 n-3 and 22:6 n-3 were observed in control diet. Palm oil substitution for fish oil increased total Saturated Fatty Acid (SFA) content of diets.
The liver of fish fed control group were composed of basophilic polygonal cells
with a spherical nucleus in the center and showed no histopathological changes
in the tissues tested by light microscope (Fig. 1a). The intestinal
wall of this group consisted of mucosa, lamina propria, muscularis and serosa
did not reveal any histopathological changes (Fig. 1b). Few
inflammatory cells, lipid vacuoles and migrated nuclei were observed in the
liver of fish fed sunflower oil diets (Fig. 2a, c).
Degenerated hepatocytes and minor hepatic steatosis were also observed in fish
fed palm oil diets (Fig. 2d). However, regenerated liver cells
that tend to exhibit a resistance to the fatty change were also observed in
fish fed these diets (Fig. 2b). Lipid vacuoles also existed
in the intestinal wall of fish fed all the vegetable oil diets (Fig.
|| Fatty acid composition (% of total fatty acid) of the experimental
||Histology of (a) Liver and (b) Intestine of fish fed with
control diet, Scale bar = 50 μm
||Histology of the liver of fish fed with vegetable oil diets,
(a) Lipid vacuoles (LV) and migration of nuclei (MN) in the liver of fish
fed with S50 diet, Scale bar = 50 μm, (b) Minor hepatic steatosis in
the liver of fish fed with P50 diet, Liver cells with minimal steatosis
in the bottom half of this photograph represent regenerated liver hepatocytes
that tend to exhibit a resistance to the fatty change, Scale bar = 200 μm,
(c) Minor hepatic steatosis in the liver of fish fed with S100 diet, Scale
bar = 50 μm and (d) Degenerated Hepatocytes (DH) in the liver of fish
fed P100 diet, Scale bar = 50 μm
|| Lipid vacuoles (LV) in the intestinal wall of fish fed with
S50 diet, Scale bar = 50 μm
Fish oil derived from industrial fisheries has been the only practical source
of EFA in the aquafeed production since many decades ago. Global fisheries are
stagnating now while the demand for fish oil is increasing along with the development
of aquaculture industry (Sargent et al., 2002).
This paradox can limit the development of aquaculture production in the future.
Therefore, finding a feasible alternative for fish oil is urgently needed in
aquafeed industry. It is now completely clear that fish oil can be partially
or fully substituted by vegetable oil in the diet of some freshwater species
without any adverse effect on their growth performance (Ng
et al., 2003). It has been also shown that substitution of fish oil
with palm oil in the diet of Malaysian mahseer improves the growth performance
of juveniles (Kamarudin et al., 2011).
On the other hand, fish oil replacement usually decreases the n-3 LC-PUFA content
of muscle and reduces the nutritional value of fish for human consumption. Therefore,
the most important limiting factor in replacing fish oil in the diet of freshwater
teleost is reduction of n-3 LC-PUFA in the muscle of fish fed fish oil free
diets. Although freshwater species are theoretically able to produce n-3 LC-PUFA
from the 18:3 n-3, most of them cannot desaturate and elongate large quantity
of 18:3 n-3. As an illustration, rainbow trout fed a 22% linseed oil diet can
only convert 12.4% of the dietary 18:3 n-3 to longer and more unsaturated homologues,
while 58.1% of this fatty acid is accumulated and 29.5% is oxidized (Turchini
and Francis, 2009). Therefore, maximum efforts have to be made to retain
the n-3 LC-PUFA in the body of farmed fish and to prevent its oxidation. Ramezani-Fard
et al. (2012) suggested that only 2.5% n-3 PUFA in the diet of T.
tambroides with adequate amount of saturated fatty acid provide the best
growth performance and retain the n-3 content of tissues.
It should be taken into account that high inclusion of vegetable oil in the
diet of fish in order to provide sufficient amounts of saturated fatty acids
may have negative effects on the fish liver. Minor hepatic steatosis was observed
in this study when fish fed a diet with partial fish oil substitution. Most
of vegetable oils, sunflower oil in particular, contain high levels of 18:2
n-6. This fatty acid has a lipogenic effect and it has been shown that its high
percentage causes intense steatosis in the most fish hepatocytes (Caballero
et al., 2004). However, the high level of 18:3 n-3, which is similar
to n-3 LC-PUFAs in term of hypolipidemic effect, does not histologically change
the hepatocytes cells (Caballero et al., 2004).
The pathogenic effects of steatosis in fish have not been fully understood.
Mosconi-Bac (1990) emphasized the pathogenic alteration
made to liver by steatosis while (Caballero et al.,
2004) considered steatosis as a physiological adaptation to the diet which
can be reversed after fish are fed a balanced diet.
In conclusion, this study showed that replacement of fish oil with vegetable oil increases lipid vacuoles in the epithelial cells of intestine and in the hepatocytes of Malaysian mahseer. Moreover, such replacement causes minor steatosis in this fish. The effects of these alterations on the growth performance and survival rate of this fish in a long time rearing period should be further investigated.
This study was supported through a Malaysian government E-Science grant No. 05-01-04-SF0209.
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