PUNJAB AGRICULTURAL UNIVERSITY

Synopsis of Thesis Problem of Post-Graduate Student: Ph.D.

Name : SONU Admission No. : L-2010-BS-72-D

Major Subject : Zoology Minor Subject : Biochemistry

Major Advisor : Dr. (Mrs.) G.K. Sehgal

1. Title of the research problem

EFFECTS OF SOME ALTERNATIVE DIETARY LIPID SOURCES ON FATTY ACID PROFILES

AND PROXIMATE COMPOSITION OF COMMON CARP, Cyprinus carpio (Linn.).

2. Introduction

Demand for fish is constantly increasing as humans are becoming more and more health

conscious. They prefer to consume nutritious food with added health benefits. Fishes possess both these

qualities and are, therefore, considered as health or functional food. Fishes are the best source of long-

chain (LC) polyunsaturated fatty acids (PUFAs), mainly the n-3 and n-6 PUFAs. The n-3 PUFAs are

known to be cardio-protective (Sanderson et al, 2002), anti-atherosclerotic (Givens et al, 2006), anti-

thrombic (Calder, 2004) and anti-arrythmatic (Givens et al, 2006). Besides, they have high protein

content (15-25%), which is required for maintenance and growth of human body. Fishes are rich in

vitamins A, D, E and K and minerals (calcium, phosphorus and iron). Compared to beef, mutton and

chicken, fish meat is more digestible as it contains much less connective tissue (Calder, 2004).

The increased demand of fish can be met from aquaculture as the capture fisheries is towards

decline. World capture fisheries decreased from 92.4 million metric tonnes (mmt) in 2004 to 90 mmt in

2009. The world aquaculture production, on the other hand, markedly increased from 15.3 mmt in 2004 to

21 mmt in 2009 (FAO, 2010). Therefore, aquaculture, with per capita supply increasing from 0.7 kg in

1970 to 7.8 kg in 2008, with an average annual growth rate of 6.6% (FAO, 2010), is the only hope.

However, aquaculture largely depends upon capture fisheries for fish meal and fish oil used in aquafeeds.

Fish oil has a high level of n-3 highly unsaturated fatty acids (n-3 HUFA), particularly, eicosapentaenoic

acid (EPA) and docosahexaenoic acid (DHA), which have high health value for fish and human beings.

Since aquaculture is expanding and capture fisheries is contracting, the supply of fish meal and fish oil is

becoming limited and hence more expensive. It has created pressure on the aquafeed manufacturers to

1

replace these ingredients with some sustainable alternatives (Pickova & Morkore, 2007). Vegetable oils

have potential to replace substantial amount of fish meal/fish oil in the diets of many fish species without

affecting their growth and feed efficiency. However, their effect on nutritional value of fish in terms of

fatty acid profiles and proximate composition of flesh is largely unknown, although oils derived from

unicellular algae, pelagic organisms or benthic invertebrates containing high amounts of n-3 HUFA have

been identified and tested in aquafeeds (Hertrampf & Piedad-Pascual 2000, Carter et   al, 2003 and Olsen

et   al, 2004 ). Nevertheless, their prices are still too high to be commonly used in aquafeeds (Turchini et al,

2009).

Thus, it is important to study the impact of vegetable oils and animal fats on the growth

performance and, more importantly, on the fatty acid and proximate compositions of the edible part of the

fish fed these oils/fats. Realizing the need for the use of vegetable oils and/or animal fats as fish feed

ingredients, some work in this direction has been done quite recently. The fish species which have been

studied include rainbow trout (Brown et al 2010, Guler and Yildiz 2011 and Trushenski et al, 2011a),

cobia (Trushenski et al, 2011c), pike perch (Kowalska et al, 2011), Oncorhynchus mykiss (Twibell et al

2011), Atlantic salmon (Menoyo et al 2005), Huso huso (Hosseini et al 2010), Litopenaeus vannamei

(Gonzalez-Felix et al 2010). No such study has been done on carps, which are important freshwater food

fishes forming about 86% of the Asian aquaculture, which contributes more than 85% to world fish

production. The present study is therefore, proposed to identify a vegetable oil and/or animal fat which is

capable of completely or partially replacing fish meal/fish oil without compromising with nutritional

quality and fatty acid composition of common carp, Cyprinus carpio (Linn.), an important freshwater

food fish.

Knowledge gaps

There is scanty information on the alternative lipid sources including vegetable oils/animal fats

which can replace fish meal/fish oil from aquafeeds. This information is completely lacking with respect

to carps, which are one of the important freshwater food fishes cultured in Asia, India and Punjab.

Objective

To identify alternative lipid source(s) for complete/partial replacement of fish meal/fish oil from

the feed of common carp without compromising with the growth performance, and nutritional quality

(fatty acid profiles and proximate composition) of the fish.

2

3. Expected new knowledge

New knowledge on whether or not vegetable oils and/or animal fat can completely/partially

replace fish meal/fish oil from carp feed, without affecting its growth performance and nutritional quality

(fatty acid profiles and proximate composition), shall be generated.

4. Review of literature

Studies on the use of alternative dietary lipid source and their efficiency to replace fish meal/fish

oil from aquafeeds have been conducted quite recently on non carp species.

The effects of dietary lipid source and n-3 highly unsaturated fatty acids (n-3 HUFA) level on

growth, body composition and blood chemistry of juvenile starry flounder was investigated by Lee et al

(2003). The contents of n-3 HUFA in fish linearly increased with increasing dietary squid liver oil.

Results indicated that dietary n-3 HUFA were essential and a value of at least 0.9% of n-3 HUFA in the

diet could be recommended for optimum growth and efficient feed utilization of juvenile starry flounder.

The twin problems of fish oil (FO) replacement with vegetable oil (VO) and high energy diets in

salmon throughout the entire production cycle was investigated Tocher et al (2003). Fatty acid

compositions of liver, intestinal tissue and gill were altered by the diets with increased proportions of C18

polyunsaturated fatty acids and decreased proportions of n-3 HUFA in fish fed VO compared to those fed

FO, HUFA synthesis in hepatocytes and enterocytes was significantly higher in fish fed VO, whereas β-

oxidation was unaltered by either of the dietary oil content or type. Over the entire production cycle,

HUFA synthesis in hepatocytes showed a decreasing trend with age, interrupted by a large peak in

activity at seawater transfer. Gill cell prostaglandin (PG) production showed a possible seasonal trend,

with peak activities in winter and low in summer and at seawater transfer. The PG production in seawater

was lower in fish fed the high-oil diets with the lowest PG production generally observed in fish fed high

VO. The changes in fatty acid metabolism induced by high dietary oil and VO replacement contributed to

altered flesh lipid content and fatty acid compositions. Similarly, Turchini et al (2003) evaluated the

effects of alternative dietary lipid sources (fish oil as control—C; canola oil—CO; poultry fat—PF; pork

lard—PL; and oleine oil—OO) on performance, tissue chemical composition, mitochondrial fatty acid

oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L.) over 70 days at

14.6±0.4 °C. The best growth was observed in fish fed the C diet whereas the PL diet fed fish had the best

feed utilization. The fatty acid composition of muscle largely reflected that of the diets, while total

cholesterol was not affected. This study showed that alternative lipid sources could be used effectively for

oil coating extruded diets for brown trout.

Berge et al (2004) examined effects of different levels of dietary conjugated linoleic acid (CLA)

on the growth and body composition of 0.7 g Atlantic salmon fry. Groups of fish were fed diets

3

containing 0%, 0.5%, 1.0% or 2.0% CLA for a period of 12 weeks. CLA did not have a significant effect

on the growth rate or on the proximate composition of salmon fry, even though there was a trend towards

higher final weight in the fish fed the 1% CLA diet. The fatty acid composition was strongly affected by

diet. CLA was deposited in the fish. Dietary CLA also affected the deposition of other fatty acids. The

deposition ratio (RD) values of 14:0, 16:0 and 18:0 fatty acids increased, while those of 16:1 and 18:1

fatty acids decreased, in response to increasing levels of CLA in the diets. This suggests that CLA causes

a reduction in D-9 desaturase activity. Dietary CLA caused a significant increase in the RD values of total

n-3 fatty acids, especially that of 22:6n-3. CLA also caused a higher concentration of phosphorus (P) and

calcium (Ca), and a lower P/Ca ratio in the fish. The results showed that dietary CLA may alter fatty acid

metabolism and bone mineralization.

Suitability of two plant based lipid sources, canola oil (CO) and linseed oil (LO), as alternatives

to fish oil for juvenile Murray cod was determined by Francis et al (2006). The fatty acid composition of

Murray cod fillet was reflective of the dietary lipid source. Fillet of fish fed the FO had highest EPA,

Arachidonic acid (ArA) and DHA. Fish fed the CO diet had high concentrations of oleic acid (OlA)

(192.2±10.5 mg g−1lipid), while the fillet of Murray cod fed the LO diet was high in α-linolenic acid

(LnA) (107.1±6.7 mg g lipid−1). This suggested that FO can be replaced by up to 100% with canola oil

and by up to 50% with linseed oil in Murray cod diets with no significant effect on growth.

An experiment in which fillets of trout fed a diet containing either 200 (low vitamin E [LVE]

diet) or 5000 (high vitamin E [HVE] diet) mg a-tocopheryl acetate/kg for 0, 4, and 9 weeks was

conducted by Jittinandana et al (2006). These fillets were evaluated fresh and after 6 months of frozen

storage. Frozen fillets were thawed and stored for 3 days at 1 °C before analyses. Muscle -tocopherol of

fish fed the HVE diet continuously increased through 9 weeks of feeding. Reduced muscle -tocopherol

and moisture, and increased muscle redness and fat were observed in frozen-refrigerated fillets compared

with fresh fillets. Thiobarbituric acid-reactive substances were lower in frozen-refrigerated fillets

produced from fish fed the HVE diet. Proportion of unsaturated fatty acids and omega-3 fatty acids

increased as feeding duration increased from 0 to 9 week.

Kleveland et al (2006) investigated effects of in vivo administration of 3-thia fatty acids (FAs) on

lipid metabolism in muscle and liver of Atlantic salmon. The fish were fed fish meal (FM) and fish oil

(FO) - based diets supplemented with either nothing (FO), or 0.3% and 0.6% of the 3-thia FAs

dodecylthioacetic acid (DTA) and tetradecylthioacetic acid (TTA) respectively. None of the 3-thia FA

diets affected lipid content of the salmon muscle. The liver index was significantly higher and the total

liver fat content was lower in the TTA group than in the FO group. Both DTA and TTA were

incorporated into the lipid fraction of muscle and liver (0.4% to 0.9%). There were no major differences

4

in the total FA composition of liver and muscle between the dietary groups; except for a small increase of

n-3 PUFAs in liver of the DTA group.

Effect of diets enriched with Δ6 desaturated fatty acids (18:3n−6 and 18:4n−3), on growth, fatty

acid composition and HUFA synthesis in two populations of Arctic charr (Salvelinus alpinus L.) was

examined by Tocher et al (2006). Dietary Echium oil (EO) had no detrimental effect on growth

performance and feed efficiency, mortalities, or liver and flesh lipid contents in either population. The

proportions of 18:2n−6 (Linoleic Acid), 18:3n−3 (Alpha-Linolenic Acid), 18:3n−6 (Gamma-Linolenic

Acid), 18:4n−3 (Stearidonic Acid), 20:3n−6 (Dihomo-Gamma-Linolenic Acid) and 20:4n−3 (Arachidonic

Acid) in total lipid in both liver and flesh were increased by dietary EO in both populations. However, the

percentages of 20:5n−3 (Eicosapentanoic Acid) and 22:6n−3 (Docosahexanoic Acid) were reduced by EO

both in liver and flesh in both strains, whereas 20:4n−6 (Arachidonic Acid) was only significantly

reduced in flesh. In fish fed FO, HUFA synthesis from both [1-14C] 18:3n−3 and [1-14C] 20:5n−3 (EPA)

was significantly higher in the planktonivorous Coulin charr compared to the demersal, piscivorous

Rannoch charr morphotype. However, HUFA synthesis was increased by EO in Rannoch charr, but not in

Coulin charr. Dietary EO had differential effects in the two populations of charr, with HUFA synthesis

only stimulated by EO in the piscivorous Rannoch morphotype, which showed lower activities in fish fed

FO.

A Ten-week experiment on Japanese sea bass (5.87±0.02 g) to study the effects of replacement of

fish oil with six alternative lipid sources: pork lard, PL; beef tallow, BT; poultry fat, PF; soybean oil, SO;

corn oil, CO; and a mixed-fat (MF: tallow, 60%; soy oil, 20%; fish oil, 20%) on growth performance and

fatty acid (FA) composition in fillet and liver was conducted by Xue et al (2006). Weight gain (WG),

specific growth rate (SGR), feed conversion ratio (FCR), feed intake and hepatosomatic index (HSI) of

fish fed the experimental diets were not significantly different. Protein efficiency ratio (PER) in fish fed

the PF diet was significantly lower than in those fed SO and CO diets. Significant differences in carcass

moisture and lipid contents of carcass and liver were observed among fish with different dietary

treatments. The fatty acid composition of fish fillets and livers reflected the dietary FA composition.

Similarly, Yilmaz and Genc (2006) conducted 60 days feeding trial to determine the effect of increasing

alternative dietary lipid [soyacid oil (SAO) and yellow grease (YG)] levels on growth performance, body

composition and liver morphology of common carp. Seven isonitrogenous practical diets were formulated

to contain 4, 8.5, 13, 18% SAO and 4, 8.5, 13% YG and the control diet without supplementation with

dietary oil. Growth performance of fish fed diet containing 8.5% YG showed the best weight gain and

was similar to the control group in respect to feed conversion, daily feed intake, protein and, energy

consumption. No improvement was found in growth parameter in SAO-fed groups. In addition, liver

5

lipoid degeneration (steatosis) was observed in fish fed with the highest dietary lipid content. The results

indicated that common carp did not efficiently utilize SAO and YG as alternative dietary lipid source.

Blanchard et al (2008) evaluated the effect of different n-3 to n-6 ratios, underlying the effect of

Linoleic Acid (LA) and Lenolenic Acid (LnA) levels, on growth performance, tissue fatty acid

composition and hepatic ultrastructure of Eurasian perch. An increased proportion of 18:2n-6 (Linoleic

Acid) and 18:3n-3 (Lenolenic Acid) in the diet of Eurasian perch, or a partial substitution of fish oil by

vegetable oil, does not appear to compromise growth performance of juvenile perch. Analyses of tissue

FA profiles indicated that a minimum level of EPA and DHA has to be incorporated into the diets to

comply with the EFA requirement of this species and that the overabundance of 18:2n-6 observed in SO

(Cod liver oil/Safflower oil) fed fish, may cause an apparent deficiency in 18:3n-3. Consequently, the

quantity of 18:2n-6 and 18:3n-3 in the SLO (Cod liver oil/Safflower oil/Linseed oil) diet (0.64

LnA/LAratio) seems to be more suitable than in the SO diet (0.03 LnA/LA ratio) to promote Arachidonic

Acid (ArA) biosynthesis.

Feeding experiment using crustacean and fish as prey for the European cuttlefish Sepia offcinalis

to evaluate the effect of prey fatty acids on the fatty acid profile of this marine predator was conducted by

Fluckiger et al (2008). Cuttlefish fed a fish diet for 29 days prior to the switch in diet were comparatively

higher in 16:0 (Palmitic Acid), 20:4 ω6 (Arachidonic Acid), 20:1ω9 (Oleic Acid), 22:5 ω6 DPA6

(Docasapentanoic Acid), 22:4ω6 (Arachidonic Acid) and 22:5 ω3 DPA3 (Docasapentanoic Acid) than

those fed on crustaceans. Cuttlefish fed a crustacean diet for 29 days prior to the switch in diet were

comparatively higher in 17:1ω8 (Haptodecanoic Acid), 18:1ω9 (Oleic Acid), 18:2ω6, 18:1 7 (Cis-

Vaccenic acid), 20:5 ω3 EPA (Eicosapentanoic Acid) and 20:2ω6 (Eicosadienoic Acid) than those fed on

fish. Following a change in diet, the fatty acid profile of the cuttlefish digestive gland reflected that of the

new diet within 14 days. The results confirmed that the fatty acid profile of the cuttlefish digestive gland

clearly reflects the profile of its recent diet. On the otherhand, Ganuza et al (2008) produced two single

cell heterotrophs as alternative sources of DHA to fisheries-derived oils. Schizochytrium G13/2S or

Crypthecodinium cohnii biomasses, either homogenised or non-homogenised were tested in gilthead

seabream (Sparus aurata) microdiets. The results showed the potential of single cell heterotrophs as

alternative DHA sources for fish oil in microdiets for gilthead seabream but also point out the necessity of

EPA sources to completely replace fisheries-derived oils.

The effects of dietary canola oil (CO) level on growth, fatty acid composition and osmoregulatory

ability of juvenile fall chinook salmon (Oncorhynchus tshawytscha) was examined by Grant et al (2008).

Whole body fatty acid compositions were influenced strongly by the dietary fatty acid compositions. CO

6

was found to be an excellent and cost-effective source of supplemental dietary lipid for culture of juvenile

fall chinook salmon during freshwater residency.

Effect of different levels of dietary HUFAs on tissue fatty acid profiles and reproductive

performance in female zebrafish was studied by Jaya-Ram et al (2008). Results showed that fatty acid

profiles of liver, muscle, ovary and egg reflected profiles of the corresponding dietary treatment.

Increasing levels of dietary linseed oil lowered deposition of DHA, EPA and ArA (Arachidonic Acid) in

all tissues. Liver fatty acid profile implied increasing biosynthesis activities during feeding of low dietary

HUFA levels, which was supported by increased expression of hepatic desaturase and elongase mRNAs.

However, the increased HUFA biosynthesis activities were unable to compensate for the inferior hepatic

ArA, EPA and DHA levels of fish fed diet Linseed oil (LO). In muscle and ovary tissues, relatively lower

concentrations of ArA and EPA were also obtained with diet LO. There was no significant difference in

EPA and ArA levels in eggs, which imply accumulation of EPA and ARA in eggs. Results also showed

an increasing trend of ovarian desaturase and elongase gene expression during low dietary HUFA levels.

The study showed that female zebrafish reproduction benefits from the supply of dietary HUFA during

reproductive stages, despite possessing ability to increase transcription of desaturase and elongase in

various tissues during low dietary HUFA intake.

Miller et al (2008) conducted an experiment in which echium oil (EO) was fed to seawater

Atlantic salmon for 12 weeks and compared with fish fed a diet containing canola oil (CO), a source of

Alpha-linolenic acid (ALA) or Fish oil (FO) that provides (n-3) LC-PUFA. Gene expression of liver Fatty

Acid (FA) elongase and Fatty Acid Desaturase 5 (FAD5) was upregulated in EO fed fish compared with

FO fish. Furthermore, dietary precursors affected the FA concentrations of direct biosynthetic products in

all tissues. The increased gene expression in the EO fed fish was reflected by an increased concentration

of EPA in the liver compared with the CO fed fish. However, the high concentrations of LC-PUFA (n-3)

found in seawater Atlantic salmon fed diets rich in FO were not attained via biosynthesis from precursors

(ALA or Stearidonic acid, SDA) in diets.

The impact of dietary replacement of fish oil by vegetable oils (VO) on gilthead seabream

(Sparusaurata)’s growth performance, nutritient utilization, body composition, and fatty acid profile was

determined by Wassef et al (2009). Consumption of VO for 20 weeks did not significantly alter the major

nutrient composition of fish, but the muscle fatty acid profile was significantly altered compared to the

reference fish oil (FO) diet fed fish. Comparatively reduced levels of EPA and DHA, as well as elevated

levels of Lenoleic acid (LA) and lenolenic acid (LNA) compared with fish fed the FO were noticed.

Similarly, Gonzalez-Felix et al (2010) evaluated replacement of marine fish oil (MFO) with alternative

oils in a plant based diet. Litopenaeus vannamei juveniles (1.55 g) were stocked into 650 L circular tanks

at 26 shrimp tank−1 and fed 13 experimental diets over a 58-day growth trial. The results showed no

7

statistically significant differences in final mean weight, growth, survival or FCR values of shrimp fed

various diets. Fatty acid (FA) profiles of tail muscle from shrimp fed the various lipid sources in general

conformed to the lipids of the feed. Shrimp fed diet 11, with 19.81 mg of linolenic acid per gram of diet

had the highest amount of this FA in shrimp tail muscle (5.61 mg g−1 wet tissue) and a relatively high n-

3/n-6 ratio of 1.15, but at the same time, practically the lowest content of EPA (4.07 mg g−1 wet tissue)

and DHA (2.04 mg g−1 wet tissue) among the dietary treatments.

Dietary fatty acid deposition was affected by the time of feeding, and hence identified possible

innovative feeding strategies towards more efficient use of dietary fish oil. Over a period of 12 weeks,

three diets with different lipid sources, canola oil (CO), fish oil (FO) or a 50/50 blend of the two oils

(Mix), were alternated daily and fed to rainbow trout (Oncorhynchusmykiss). Six treatments were

administered to fish, reference treatment (REF, continuously fed FO), control treatment (CT,

continuously fed Mix), am canola oil ration (amCOR), pm canola oil ration (pmCOR), am canola oil

satiation (amCOS) and pm canola oil satiation (pmCOS). Fish received either the CO diet in the am or pm

feeds and those received the FO diet at the opposite time. A significant increase in growth and feed

consumption was noted in the pmCOS treatment. Fillet fatty acid profile was modified by associated

feeding schedules and was generally reflective of dietary fatty acid profile. No significant increases in

n−3 LCPUFA deposition were observed. However, both LA and ALA contents were significantly higher

in pmCOR compared to amCOR and CT. The results suggested existence of cyclical circadian patterns in

fatty acid deposition in rainbow trout (Brown et al, 2010).

Hosseini et al (2010) investigated effect of dietary alpha-tocopheryl acetate (vitamin E) and oil

sources on fish flesh quality characteristics of Huso huso during frozen storage. Practical-type diets

containing 0 or 250 mg vitamin E kg−1 with three lipid sources, fish oil (FO), soybean oil (SO) and canola

oil (CO), were fed to H. huso for 120 days. Fillet samples were analysed fresh or after storage at

−18 ± 1 °C for 12 months. Replacement of FO by SO and CO in diets for H. huso significantly altered the

fatty acid (FA) profile, which also influenced the FA composition during frozen storage. Dietary vitamin

E had a significant effect on muscle vitamin E content and lipid oxidation during storage. Similarly, Lim

et al (2010) determined the effect of increasing dietary levels of fish oil on vitamin E requirement and

their effect on growth performance, liver vitamin E status, and tissue proximate and fatty acid

compositions of channel catfish. Weight gain, feed intake, and feed efficiency ratio were not affected by

dietary levels of fish oil, vitamin E, or their interaction. Whole-body moisture significantly decreased and

lipid increased when dietary lipid levels were increased to 10 or 14%. Dietary vitamin E levels had no

effect on body proximate composition. Lipid content of liver was not influenced by dietary levels of fish

oil and vitamin E or their interaction. Fatty acid composition of whole body and liver reflected that of

dietary lipid but was not influenced by dietary levels of vitamin E. Whole-body saturates increased,

8

whereas MUFAs decreased with increasing dietary levels of fish oil. Liver saturates were not affected by

fish oil levels, but MUFAs and n-6 decreased and increased, respectively, with increasing fish oil levels.

Total n-3 and n-3 HUFA in both the tissues increased with increasing fish oil levels in diets, but liver

stored much higher levels of these fatty acids.

The effect of different dietary Alfa Linolenic Acid (ALA) to Linoleic Acid (LA) ratios, while

employing 100% fish oil deprived diets, on growth performance and flesh fatty acid composition of

Murray cod was evaluated by Senadheera et al (2010). Results showed that an increased dietary ALA/LA

ratio did not impair growth performance or the tissue lipid concentration of the fish. The dietary ALA/LA

ratio had significant impact on the final fatty acid make-up and nutritional quality of the fish fillet. In

particular, the fillets of fish fed higher ALA/LA ratios (hence receiving more dietary ALA) recorded

significantly higher concentrations of EPA and DHA. High dietary LA content, however, was shown to

have negative impacts on the efficiency of a finishing strategy. Only 10% of the total EPA and DHA

provided during the finishing period via the fish oil-based diet was retained and deposited into the fish

fillet. The deposition of EPA and DHA during the finishing period was shown to be affected by previous

feeding history. Fish previously fed high LA diets deposited significantly lower amounts of these fatty

acids in comparison to the fish previously fed a diet rich in ALA.

Babalola et al (2011) investigated the influence of fish oil (FO), two terrestrial animal fats, pig

lard (PL) and poultry fat (PF) and three vegetable oils [palm kernel oil (PKO), sheabutter oil (SBO) and

sunflower oil (SFO)] as the dietary lipid sources on the growth performance, nutrient digestibility, fatty

acid (FA) composition and histology of Heterobranchus longifilis. The lipid source influenced weight

gain, specific growth rate, feed conversion ratio, epatosomatic index and nutrient digestibility. The results

of this study show that alternative lipid sources could be used in H. longifilis diets. However, inclusion of

PKO or SBO in the diet of H. longifilis produced fillets with low concentrations of long chain n-3 PUFA.

Moreover, the effects of dietary fish oil replacement by cottonseed oil on growth performance and fatty

acid composition of rainbow trout (Oncorhynchus mykiss) was investigated by Guler and Yildiz (2011).

Fillet fatty acid composition reflected dietary fatty acid composition. The n-6 PUFA concentration

increased with increasing cottonseed oil levels in the diets. In contrast, the n-3 PUFA levels decreased

with increasing cottonseed oil levels in the diets. The highest level of EPA and DHA concentrations were

recorded in fish fed the FO diet and the lowest in those fed 100% cottonseed oil diet. And Gumus (2011)

examined the effect of replacement of fish meal (FM) in diets with sand smelt meal (SSM) on fatty acid

composition of carp fry, Cyprinus carpio. Fatty acid analysis showed that saturated fatty acids in fish

muscle significantly decreased, but MUFA and PUFA did not change with increasing dietary SSM. The

amounts of 15:0 (Pentadecanoic Acid), 17:0 (Haptodecanoic Acid), 18:1n-7 (cis-Vaccenic Acid), 18:2n-6

(Linoleic Acid) and 22:5n-3 (DPA) significantly increased, but 16:0 (Palmitic Acid), 18:1n-9 (oleic

9

Acid), 18:3n-3 (Linolenic Acid) and 20:1 n-9 (Eicosenoic Acid) significantly decreased with increasing

dietary SSM. Total n-6 PUFA increased with increasing dietary SSM, but total n-3 PUFA were not

changed in muscle of fish fed the experimental diets. The ratio of n-3 to n-6 was not affected significantly

in muscle of fish fed the experimental diets containing different proportions of SSM, including the control

diet.

Preliminary feeding trials with goldfish Carassius auratus by using four practical diets with 4%

or 10% supplemental poultry fat (PF) and 0% or 2% dairy–yeast prebiotic was conducted by Lochmann et

al (2011). In pools, weight gain was higher in goldfish that received the 10% PF diets or the 4% PF diet

with prebiotic than in fish that were fed the 4% PF diet without prebiotic. Lipid and dry matter were

higher in goldfish that were given 10% PF diets than in fish that were given 4% PF diets. Subsequent

bacterial challenges with Flavobacterium columnare were conducted separately for fish in pools that

received the 4% PF and 10% PF diets. Results indicated that the dairy–yeast prebiotic has some potential

to protect stressed goldfish against F. columnare infection.

Whether the sequence of dietary LC-PUFA provision affected tissue composition of sunshine

bass and whether profile change was similar after a switch to or from LC-PUFA-rich feed was

investigated by Trushenski et al (2011b). Sunshine bass were fed practical feeds (~45% protein, ~14%

lipid) containing FO (rich in LC-PUFAs) or corn oil (CO; rich in medium-chain [MC] PUFAs). Feeding

was done according to four regimens: FO feed exclusively, CO feed exclusively, or switching from one

feed to the other halfway through the trial (from CO to FO or from FO to CO). Administering the FO and

CO feeds exclusively or in alternation did not significantly affect production performance. Fillet FA

profile changed little for fish that received the exclusive FO and CO regimens and essentially became

slightly more FO-like and CO-like, respectively. Fillet LC-PUFA and MC-PUFA levels were comparable

in the alternating FO-to-CO and CO-to-FO groups at harvest. Trushenski et al (2011a) also conducted an

experiment in which rainbow trout Oncorhynchus mykiss were reared on feeds containing FO or a 50:50

blend of FO and coconut oil (COCONUT), palm oil (PALM), standard soybean oil (STD-SBO),

hydrogenated soybean oil (HYD-SBO), low-18:3(n-3) (alpha-linolenic acid) soybean oil (LOALA-SBO),

or low-18:3(n-3) canola oil (LO-ALA-CAN). Two saturated fatty acids (SFA)-enriched lipids derived

from the processing of cottonseed (SFA-COT) or soybean (SFA-SBO) was also evaluated as 50% FO

substitutes. After 7 weeks, growth performance was largely unaffected by dietary lipid source. Fillet

levels of long-chain (LC) polyunsaturated FAs (PUFAs) among fish that received the HYD-SBO, LO-

ALA-SBO, SFA-SBO, and SFA-COT feeds were equivalent to levels in fish that received the FO feed,

despite an approximate 50% reduction in dietary LC-PUFA intake. The results indicated that feeds

containing a blend of FO and novel soy- or cottonseed-derived lipids yielded equivalent growth

10

performance and fillet LC-PUFA content in rainbow trout. The use of STD-SBO, COCONUT, PALM, or

LO-ALA-CAN did not impair growth efficiency but did alter the fillet FA profile.

A 12-week feeding trial to evaluate a fish-meal-free, fish-oil-free diet for use with first-feeding

steelhead, Oncorhynchus mykiss was conducted by Twibell et al (2011). The marine-based control diet

(marine diet) contained sardine (Sardinops spp.) meal and pollock (Pollachius virens) liver oil as the

primary sources of protein and lipid, respectively. The experimental diet (terrestrial diet) contained only

terrestrial sources of protein (poultry by-product meal, blood meal, canola, corn gluten, and wheat gluten)

and lipid (canola oil and flaxseed oil). After 2 weeks of feeding, Steelhead that received the marine diet

exhibited significantly higher carcass concentrations of 14:0 (Tetradecanoic Acid), 16:0 (Hexadecanoic

Acid), 16:1 (Palmitoleic Acid), 20:1(Eicosenoic Acid) , 20:5(n-3) Eicosapentanoic acid (EPA), 22:5(n-3)

Docosapentanoic Acid (DPA), and 22:6(n-3) (DHA) fatty acids but significantly lower carcass

concentrations of 18:1(cis-Vaccenic Acid) , 18:2(n-6) (Linoleic Acid), 18:3(n-6) (GLA), 18:3(n-3)

(ALA), and 20:4(n-6) (Arachidonic Acid) relative to fish that were given the terrestrial diet.

The extent to which flax oil can replace fish oil in diets fed to female broodstock of white bass,

Moronechrysops was determined by Lewis et al (2011) by evaluating growth performance and the fatty

acid (FA) profiles of ovum lipid classes. Results indicated that flax oil has potential for use as an

alternative to menhaden fish oil in diets for female white bass broodstock without altering phospholipid

LC-PUFA content. Similarly, Menoyo et al (2005) studied effect of dietary fish oil substitution with

linseed oil (LO) on growth performance, tissue fatty acid profile, metabolism, and oxidative stability of

Atlantic salmon and concluded that LO can totally replace FO in Atlantic salmon feed without affecting

growth performance and muscle susceptibility to lipid oxidation. Fatty acid metabolism was affected by

LO, promoting glucose-6-P-dehydrogenase (G6PD) activity and eicosatetraenoic acid accumulation;

however, a 100% LO replacement decreased concentrations of EPA and DHA in salmon muscle.

Effects of dietary n-3 highly unsaturated fatty acid (HUFA) concentrations on spawning

performance and fatty acid composition of broodstock, eggs and larvae of Acanthopagruslatus was

investigated by Zakeri et al (2011). The results showed that the n-3 HUFA concentrations of lipids in

broodstock diet had a considerable effect on the quality and fatty acids composition of egg and larvae in

A. latus.

IPR search has been made on the following sites:

1. www.patentoffice.nic.in/ipr/patent/patents.html

2. www.google.com/patents

3. www.getthepatent.com

11

4. www.freepatentsonline.com

5. Technical Programme

Experiment 1

i. Name of the experiment: Study on fatty acid profiles of some alternative dietary lipid

sources.

ii. Location/place of work: Department of Zoology, College of Basic Sciences and Humanities,

Punjab Agricultural University, Ludhiana.

iii. Methodology: Replicated samples of some alternative dietary lipid sources including 3

vegetable oils (soybean oil, canola oil and sunflower oil) and two animal fats (poultry fat and

mutton fat) will be analyzed for their fatty acid profiles by Gas Chromatography.

iv. Observations to be recorded: Observations will be made on fatty acid profiles of the

alternative dietary lipid sources mentioned above following the method of AOAC (2000).

v. Statistical analysis: The data will be analyzed by ANOVA to determine the significance of

differences in the fatty acid composition of the alternative lipid sources.

Experiment 2

i. Name of the experiment: Effects of some alternative dietary lipid sources on growth

performance, fatty acid profiles and proximate composition of common carp, Cyprinus carpio

(Linn.).

ii. Location/place of work: Department of Zoology, College of Basic Sciences and Humanities,

Punjab Agricultural University, Ludhiana.

iii. Methodology: Effects of each alternative dietary lipid source (soybean oil, canola oil, sunflower

oil, poultry fat and mutton fat) will be studied as given below:-

a) Treatments- Five dietary treatments (0%, 25%, 50%, 75% and 100% replacement

of fish meal/fish oil with an alternative lipid source).

12

b) Replications- three.

c) Rearing period- 60 days.

iv. Observations to be recorded: The observations will be made on:

a) Water quality in terms of pH, DO, temperature, alkalinity, salinity, ammonia and total

hardness, on weekly basis. Analysis will be done as per the standard methods of APHA

(1991).

b) Growth performance in terms of net weight gain, average daily growth and specific

growth rate, on weekly basis.

c) Fatty acid profiles and proximate composition of the fish fed different experimental diets

on zero day and after 60 days of feeding by following the methods of AOAC (2000).

v. Statistical analysis: Analysis of variance (ANOVA) technique will be applied to determine the

significance of differences in the water quality parameters, growth performance, and fatty acid

profiles of the fish fed different experimental feeds.

Experiment 3

i. Name of the experiment: Study on finishing effect of fish meal/fish oil on growth

performance, fatty acid profiles and proximate composition of Cyprinus carpio (Linn.).

ii. Location/place of work: Department of Zoology, College of Basic Sciences and Humanities,

Punjab Agricultural University, Ludhiana.

iii. Methodology:

a) Treatments- three

(i) Feeding fish with diet containing the vegetable oil (that resulted in best fatty

acid profile) for the first 40 days and the one containing only fish meal/fish

oil for the remaining 20 days of the 60 day feeding.

(ii) Feeding fish with diet containing the animal fat (that resulted in best fatty

acid profile) for the first 40 days and the one containing only fish meal/fish

oil for the remaining 20 days of the 60 day feeding.

(iii) Feeding fish with the diet containing only fish meal/fish oil for 60 days.

b) Replications- three.

c) Rearing period- 60 days.

iv. Observations to be recorded: As in Experiment No. 2.

v. Statistical analysis: As in experiment 2

13

6. Schedule Programme of Work

Sr. No.

Activity Semester II

(2011)

Semester III

(2012)

Semester IV

(2012)

Semester V

(2013)

Semester VI

(2013)

J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D

I Collection of relevant literature

★ ★

Preparation and submission of synopsis

★

II Procurement of common carp, materials for research, standardization of methods for the estimation of water quality, fatty acid profiles, proximate composition and handling, rearing, feeding and sampling of common carp

★ ★ ★

III Experiment No. 1 and samples analyses

★ ★

IV Experiment No. 2 and samples analyses

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

V Experiment No. 3 and samples analyses

★ ★ ★

VI Data collection and compilation

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

Statistical analysis

★ ★

VII Thesis writing ★ ★ ★

Rough thesis submission

★

Final thesis submission

★

JFMA…. D refers to name of the month.

7. Collaboration with other departments: Nil

14

8. References

AOAC (2000) Official Methods of Analysis (17th Edition). Meat and meat products Ch. 39, pp: 3. 481 North Frederick Avenue Gaithersburg, Maryland 20877-2417 USA.

APHA (1991) Standard methods for the examination of water and waste water (18 th Edition). pp 1193. American Public Health Association, Washington D. C.

Babalola TO, Apata DF, Omotosho JS and Adebayo MA (2011) Differential Effects of Dietary Lipids on Growth Performance, Digestibility, Fatty acid composition and histology of African catfish (Heterobranchus longifilis) fingerlings. Food and Nutrition Sciences 2, 11-21.

Berge GM, Ruyter B and Asgard T (2004) Conjugated linoleic acid in diets for juvenile Atlantic salmon (Salmo salar); effects on fish performance, proximate composition, fatty acid and mineral content. Aquaculture 237: 365–80.

Blanchard G, Makombu JG and Kestemont P (2008) Influence of different dietary 18:3n-3/18:2n-6 ratio on growth performance, fatty acid composition and hepatic ultrastructure in Eurasian perch, Perca fluviatilis. Aquaculture 284: 144–50.

Brown TD, Francis DS, Turchini DM (2010) Can Dietary Lipid Source Circadian Alternation Improve omega-3 Deposition in Rainbow Trout? Aquaculture 300: 148-155.

Calder PC (2004) n-3 Fatty acids and cardiovascular diseases: evidence explained and mechanisms explored. Clinical Sci 107: 1-11.

Carter CG, Bransden MP, Lewis TE, Nichols PD (2003) Potential of Thraustochytrids to partially replace fish oil in Atlantic salmon feeds. Marine Biotechnology 5: 480–492.

FAO (2010) The State of World Fisheries & Aquaculture www.fao.org/docrep/013/i1820e/i1820e01.pdf (Accessed on 17.08.2011).

Fluckiger M, Jackson GD, Nichols P, Virtue P, Daw A and Wotherspoon S (2008) An experimental study of the effect of diet on the fatty acid profiles of the European Cuttlefish (Sepia officinalis). Mar Biol 154:363–372.

Francis DS, Turchini GM, Jones PL and De Silva SS (2006) Effects of dietary oil source on growth and fillet fatty acid composition of Murray cod, Maccullochella peelii peelii. Aquaculture 253: 547–56.

Ganuza E, Benítez-Santana T, Atalah E, Vega-Orellana O, Ganga R and Izquierdo MS (2008) Crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to fisheries-derived oils from seabream (Sparus aurata) microdiets. Aquaculture 277: 109–116.

Givens DI. Kliem KE and Gibbs RA (2006) The role of meat as a source of n-3 polyunsaturated fatty acids in the human diet. Meat Sci 74: 209-18.

González-Félix ML, da Silva FSD, Davis DA, Samocha TM, Morris TC, Wilkenfeld JS, Perez-Velazquez M (2010) Replacement of Fish Oil in Plant Based Diets for Pacific White Shrimp (Litopenaeus vannamei). Aquaculture 309: 152-158.

15

Grant AAM, Baker D, Higgs DA, Brauner CJ, Richards JG, Balfry SK and Schulte PM (2008) Effects of dietary canola oil level on growth, fatty acid composition and osmoregulatory ability of juvenile fall chinook salmon (Oncorhynchus tshawytscha). Aquaculture 277: 303-12.

Guler M and Yildiz M (2011) Effects of dietary fish oil replacement by cottonseed oil on growth performance and fatty acid composition of rainbow trout (Oncorhynchus mykiss).Turk. J Vet Anim Sci 35(1): 157-67.

Gumus E (2011) Fatty acid composition of fry mirror carp (Cyprinus carpio) fed graded levels of sand smelt (Atherina boyeri) meal. Asian-Aust J Anim Sci 24 (2): 264 – 71.

Hertrampf JW and Piedad-Pascual F (2000) Handbook on Ingredients for Aquaculture Feeds. Kluwer Academic Publishers, Dordrecht.

Hosseini SV, Abedian-KenariA, Rezaei M, Nazari MR, Feás X and Rabbani M (2010) Influence of the in vivo addition of alpha-tocopheryl acetate with three lipid sources on the lipid oxidation and fatty acid composition of Beluga sturgeon, Huso huso, during frozen storage. Food Chemistry 118: 341-48.

Jaya-Ram A, Kuah MK, Lim PS, Kolkovski S and Shu-Chien AC (2008) Influence of dietary HUFA levels on reproductive performance, tissue fatty acid profile and desaturase and elongase mRNAs expression in female zebrafish Danioreri. Aquaculture 277: 275–281.

Jittinandana S, Kenney PB, Slider SD, Kamireddy N and Hankins JS (2006) High dietary vitamin E affects storage stability of frozen-refrigerated trout fillets.J Food Sci 71 (2): C91-C96.

Kowalska A, Zakes Z, Jankowska B and Siwicki A (2011) Substituting vegetable oil for fish oil in pikeperch diets: the impact on growth, internal organ histology, blood biochemical parameters, and proximate composition. Aquaculture Nutrition 17: e148-e163.

Kleveland EJ, Ruyter B, Vegusdal A, Sundvold H, Berge RK and Gjoen T (2006) Effects of 3-thia fatty acids on expression of some lipid related genes in Atlantic salmon (Salmo salar L.). Comparative Biochemistry and Physiology, Part B 145: 239–48.

Menoyo D, Lopez-Bote CJ, Obach A and Bautista JM (2005) Effect of dietary fish oil substitution with linseed oil on the performance, tissue fatty acid profile, metabolism and oxidative stability of Atlantic salmon. J Anim Sci 83: 2853-62.

Lee SM, Lee JH and Kim KD (2003) Effect of dietary essential fatty acids on growth, body composition and blood chemistry of juvenile starry flounder (Platichthys stellatus). Aquaculture, 225: 269-81.

Lewis HA, Trushenski JT, Lane RL and Kohler CC (2011) Differential incorporation of dietary fatty acids from flax and fish oils into lipid classes of white bass ova. North American Journal of Aquaculture, 73 (2): 212-20.

Lim C, Fdirim-Aksoy M, Shelby R, Li MH and Klesius PH (2010) Growth performance, vitamin E status, and proximate and fatty acid composition of channel catfish, Ictalurus punctatus, fed diets containing various levels of fish oil and vitamin E. Fish Physiol Biochem 36:855–66.

16

Lochmann RT, Sink TD and Phillips H (2011) Effects of dietary lipid concentration and a dairy-yeast prebiotic on growth, body composition, and survival of stressed goldfish challenged with Flavobacterium columnare. North American Journal of Aquaculture, 73 (2): 239-47.

Miller MR, Bridle AR, Nichols PD and Carter CG (2008) Increased elongase and desaturase gene expression with stearidonic acid enriched diet does not enhance long-chain (n-3) content of seawater Atlantic salmon (Salmo salar L.). J Nutrion 38: 2179-85.

Olsen RE, Henderson RJ, Sountama J, Hemre G-I, Ringø E and Melle W (2004) Atlantic salmon, Salmo salar, utilizes wax ester-rich oil from Calanus finmarchicus effectively. Aquaculture 240: 433–449.

Pickova J and Morkore T (2007) Alternate oils in fish feeds.European Journal of Lipid Science and Technology 109 (3), 256-63.

Sanderson P, Finnegan YE, Williams CM, Calder PC, Burdge GC, Wootton SA, Griffin BA, Millward DJ, Pegge NC and Bemelmans WJE (2002) UK Food standards agency alpha-linolenic acid workshop report. British Journal of Nutrition 88: 573–579.

Senadheera SPSD, Turchini GM, Thanuthong T and Francis DS (2010) Effects of dietary α-linolenic acid (18:3n−3)/linoleic acid (18:2n−6) ratio on growth performance, fillet fatty acid profile and finishing efficiency in Murray cod. Aquaculture 309: 222–30.

Tocher DR, Dick JR, MacGlaughlin P and Bell JG (2006) Effect of diets enriched in Δ6 desaturated fatty acids (18:3n−6 and 18:4n−3), on growth, fatty acid composition and highly unsaturated fatty acid synthesis in two populations of Arctic charr (Salvelinus alpinus L.). Comparative Biochemistry and Physiology, Part B 144: 245-53.

Tocher D.R. (2003). Metabolism and functions of lipids and fatty acids in teleost fish. Reviews in Fisheries Science 11(2), 107-184.

Trushenski JT, Blaufuss P, Mulligan B and Laporte J (2011a) Growth performance and tissue fatty acid composition of rainbow trout reared on feeds containing fish oil or equal blends of fish oil and traditional or novel alternative lipids. North American Journal of Aquaculture, 73 (2): 194-203.

Trushenski JT, Gause B and Lewis HA (2011b) Selective fatty acid metabolism, not the sequence of dietary fish oil intake, prevails in fillet fatty acid profile change in sunshine bass. North American Journal of Aquaculture, 73 (2): 204-11.

Trushenski JT, Laporte J and Lewis H (2011c) Fish Meal Replacement with Soy-derived Protein in Feeds for Juvenile Cobia: Influence of Replacement Level and Attractant Supplementation. Journal of the World Aquaculture Society 42, 435-443.

Turchini G.M., Torstensen B.E. and Ng W. (2009). Fish oil replacement in finfish nutrition. Reviews in Aquaculture 1(1), 10-57.

Turchini GM , Mentasti T, Frøyland L, Orban E, Caprin F, Moretti VM and Valfre F (2003) Effects of alternative dietary lipid sources on performance, tissue chemical composition, mitochondrial fatty acid oxidation capabilities and sensory characteristics in brown trout (Salmo trutta L.). Aquaculture 225, 251–267.

17

Twibell RG, Gannam AL, Ostrand SL, Holmes JSA, Poole JB (2011) Altered Growth Rates, Carcass Fatty Acid Concentrations, and Tissue Histology in First-Feeding Steelhead Fed a Fish-Meal- and Fish-Oil-Free Diet. North American Journal of Aquaculture, 73 (2): 230-38.

Wassef EA, Saleh NE and El-Hady HAE (2009) Vegetable oil blend as alternative lipid resources in diets for gilthead seabream, Sparus aurata. Aquacul tInt 17: 421–35.

www.patentoffice.nic.in/ipr/patent/patents.html

www.google.com/patents

www.getthepatent.com

www.freepatentsonline.com

Xue M, Luo L, Wu X, Ren Z, Gao P, Yu Y and Pearl G (2006) Effects of six alternative lipid sources on growth and tissue fatty acid composition in Japanese sea bass (Lateolabrax japonicus). Aquaculture 260: 206–214.

Yilmaz E and Genc E (2006) Effects of Alternative Dietary Lipid Sources (Soy-acid oil and Yellow grease) on Growth and Hepatic Lipidosis of Common Carp (Cyprinus Carpio) Fingerling: A Preliminary Study. Turkish J Fisheries and Aquatic Sciences  6: 37-42.

Zakeri M, Kochanian P, Marammazi JG and Yavari V (2011) Effects of dietary n-3 HUFA concentrations on spawning performance and fatty acids composition of broodstock, eggs and larvae in yellowfin sea bream, Acanthopagrus latus. Aquaculture 310: 388-94.

________________________

Signature of the Student

18

ADVISORY COMMITTEE

Name Designation Department Signature

Major Advisor Dr. (Mrs.) G.K. Sehgal

Associate Professor

Zoology ___________

Member Dr. H.S. Sehgal Professor &Liaison Officer

Zoology ___________

Member Dr. (Mrs.) A.K. Atwal

Senior Biochemist Plant Breeding and Genetics

___________

Member Dr. S.S. Thind Professor Food Technology ___________

Nominee of

Dean PGS

Dr. K.S. Khera Professor Zoology ___________

Forwarded five copies to the Dean, Postgraduate Studies, for approval by the Synopsis Approval

Committee.

Name Designation Department Signature

Consultant Dr. M. Javed Associate Maths, Statistics ___________

Statistician Professor and Physics

_________________________

Head of the Department

Memo No.:

Dated:

______________________

Dean, Postgraduate Studies

19