EFFECTS OF SOME ALTERNATIVE DIETARY LIPID SOURCES ON FATTY ACID PROFILES AND PROXIMATE COMPOSITION OF COMMON CARP, Cyprinus carpio (Linn.) Dissertation Submitted to the Punjab Agricultural University in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY in ZOOLOGY (Minor Subject: Biochemistry) By Sonu (L-2010-BS-72-D) Department of Zoology College of Basic Sciences and Humanities ©PUNJAB AGRICULTURAL UNIVERSITY LUDHIANA -141004 2015 CERTIFICATE I This is to certify that the dissertation entitled, ―Effects of some alternative dietary lipid sources on fatty acid profiles and proximate composition of common carp, Cyprinus carpio (Linn.).” submitted for the degree of Doctor of Philosophy, in the subject of Zoology (Minor subject: Biochemistry) to the Punjab Agricultural University, Ludhiana, is a bonafide research work carried out by Ms. Sonu (Admn No. L-2010-BS-72-D) under supervision of Dr. (Mrs.) G. K. Sehgal, Associate Professor w.e.f. 15.3.2011 to 19.5.2014 and thereafter under my supervision and that no part of this thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully acknowledged. __________________________ [Major Advisor] Dr. (Mrs.) B.K. Babbar Assistant Zoologist (Rodents) Department of Zoology Punjab Agricultural University Ludhiana -141004 CERTIFICATE II This is to certify that the dissertation entitled, “Effects of some alternative dietary lipid sources on fatty acid profiles and proximate composition of common carp, Cyprinus carpio (Linn.).” submitted by Ms. Sonu (Admn No. L-2010-BS-72-D) to the Punjab Agricultural University, Ludhiana, in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the subject of Zoology (Minor subject: Biochemistry) has been approved by Student‘s Advisory Committee along with Head of the Department after an oral examination on the same. _____________________ _______________________________ [Dr. (Mrs.) B. K. Babbar] Dr. Devinder Singh Major Advisor Professor, Department of Zoology and Environmental Sciences Punjabi University, Patiala [External Examiner] ______________________ [Dr. (Mrs.) G. K. Sangha] Head of the Department ________________________ [Dr. (Mrs.) Neelam Grewal] Dean Post Graduate Studies ACKNOWLEDGEMENTS Before getting into thick of the things, I would like to convey a few heartfelt words of my appreciations. Foremost, I pay my tribute and indebtness to ALMIGHTY and MY PARENTS for showering ultimate blessing on me and helping me to become able to present this humble contribution to the knowledge of science. Words are compendious in expressing my profound sense of gratitude to my revered Major Advisors, Dr. (Mrs.) G.K. Sehgal, Associate Professor and Dr. (Mrs.) B.K. Babbar, Assistant Zoologist (Rodents), Department of Zoology, for their thoughtful criticism, judicious guidance, unfailing courtesy, moral encouragement, affectionate appreciation, frank and friendly association and tackling a variety of hurdles with patience during the pursuance of this study. It was very proud privilege and unforgettable experience to work under the supervision of such dedicated guides. I am extremely thankful to Dr. (Mrs.) Gurinder Kaur Sangha, Head, Department of Zoology for providing necessary inputs and research facilities for this study and most importantly her valuable suggestions and guidance. I am greatly indebted to the members of my advisory committee viz. Dr. (Mrs.) J.K. Kondal, Associate Professor, Department of Zoology, Dr. (Mrs.) Sucheta Sharma, Professor, Department of Biochemistry, Dr. S.S. Thind, Professor, Department of Food Science and Technology and Dr. K.S. Khera (Dean PGS nominee) Professor, Department of Zoology, for their able guidance, constructive suggestions and meticulously scrutinizing this manuscript. Special thanks to Dr. H.S. Sehgal (Head, Dptt. of Environmental Sciences, Jammu University) and Dr. S.S. Hundal (Professor, Dpartment of Zoology) for their considerate behaviour, valuable advice and suggestions. I am very thankful to the University Grants Commission for providing the financial help through Maulana Azad National Fellowship. It is rightly said that "Every effort is motivated by ambitious and all ambitions have an inspiration behind." I owe this pride place to my Mother Sdn. Paviter Kaur, she left this world during my Ph.D. It was not easy for me to carry on my research work at that moment but my father S. Paramjit Singh’s never ending support and blessings of my mother from heaven motivated me in bringing this manuscript in a final shape. I cannot weigh my feelings with words for my dearest brother Amandeep and sister Puneet, for their motivation, encouragement, everlasting love and affection and moral support. Heartiest thanks to my friend Dr. Navjot Kaur Lail, my senior Dr. Jyotika Tiwari and my juniors Harjot Pandher, Amanjot, Neha, Navpreet, Manjeet, Ramandeep Kaur Thind, Shilpa Panjgotra and all my friends for providing ungrudging help, cheerful acknowledgment throughout my research work. The cooperative attitude of non-teaching staff and all the attendants of Zoology Department are worth appreciating. Thanks to all my well wishers who make the world special. All may not be mentioned but none is forgotten, words may be due, but thoughts remain with me. Place: Ludhiana Dated: (Sonu) Title of the Thesis : Effects of some alternative dietary lipid sources on fatty acid profiles and proximate composition of common carp, Cyprinus carpio (Linn.) Name of the Student and : Sonu Admission No. (L-2010-BS-72-D) Major Subject : Zoology Minor Subject : Biochemistry Name and Designation of : Dr. (Mrs.) B. K. Babbar Major Advisor Assistant Zoologist (Rodents) Degree to be Awarded : Doctor of Philosophy Year of award of Degree : 2015 Total Pages in Thesis : 118+Vita Name of University : Punjab Agricultural University, Ludhiana-141004, Punjab, India ABSTRACT The study has been conducted to identify alternative dietary lipid source(s) for complete/partial replacement of fish meal/fish oil from the feed of common carp, Cyprinus carpio (Linn.) without compromising with the growth performance, and nutritional quality (fatty acid profiles and proximate composition) of the fish. The alternative dietary lipid sources taken were vegetable oils viz. soybean oil (SBO), canola oil (CO) and sunflower oil (SFO) and animal fats viz. poultry fat (PF) and goat fat (GF). Treatment diets were prepared by replacing 0%, 25%, 50%, 75% and 100% fish oil with each alternative lipid source and were fed to the experimental fish for 60 days under laboratory conditions. Results revealed that substitution of fish oil with alternative dietary lipid sources in the common carp diet has no negative effects on the growth performance and muscle proximate composition. The fatty acid composition of the diet was reflected in muscle total lipid of fish. There was a significant difference in the concentration of SFAs, MUFAs and PUFAs in the muscle of C. carpio fed different experimental diets. The levels of n-3 HUFAs decreased with increasing vegetable oil/animal fat percentages in all the diets (p≤0.05). Similarly, the ratios of n-3 to n-6 fatty acid decreased with increasing vegetable oil/animal fat percentages in all the diets. As compare to vegetable oils, animal fats were found unbalanced especially in terms of fatty acid profiles due to low level of n-3 PUFAs. However 25-50% replacement of fish oil with vegetable oil/animal fat retained ratio of n-3/n-6, PUFA/SFA and PUFA/MUFA within recommended range. Considering the above point, 50% canola oil diet and 50% poultry fat diet were chosen for next experiment to study the finishing effect of 100% fish oil on the fatty acid composition of fish muscle. The results of this experiment suggest that inducing a dietary shift from canola oil based and poultry fat based feeds to fish oil based feeds supplied as finishing diet about 20 days before harvesting strongly increases long-chain PUFA concentrations in common carp as compared to those fed only canola oil and poultry fat based feeds throughout the rearing period. Key words: Cyprinus carpio, fish oil replacement, vegetable oils, animal fats, fatty acid profile, growth, proximate composition _______________________ ____________________ Signature of Major Advisor Signature of the Student Koj gRMQ dw isrlyK : isprIns kwrpIE (iln.) dI PYtI AYisf pRoPwiel Aqy pRogzImyt bxqr aupr Kurwk iv`cl y ilipf dy bdlvyN sroqW dy pRBwv dw mlu Wkx ividAwrQI dw nwm Aqy : sonU dwKlw nM. (AYl-2010-bI AYs-72-fI) pRmu`K ivSw : jIv-ivigAwn sihXogI ivSw : jIv-rswiex ivigAwn pRmu`K slwhkwr dw nwm Aqy : fw. (SRImqI) bI. ky. b`br Ahudw shwiek jIv ivigAwnI (rofYNts) ifgrI : pI.AYc.fI. ifgrI nwl snmwinq krn dw swl : 2015 Koj gRMQ iv`c ku`l pMny : 118+vItw XUnIvristI dw nwm : pMjwb KyqIbwVI XUnIvristI, luiDAwxw–141 004, pMjwb, Bwrq incoV mOjUdw AiDAYn m`CI dy ivkws Aqy pOSitk guxvqw (PYtI Ayisf pRoPweIl Aq y pRokzImyt bxqr) nwl smJOqw kIq y ibnW kwmn kwrp, isprIns kwrpIE (iln.) dI Kurwk iv`c m`CI/m`CI dy qyl dy pUrn/ApUrn bdlwA leI ilipf dy bdlvyN sroqW dI pihcwx krn leI kIqw igAw[ sbzIAW dy qyl ijvyN ik soieAwbIn dw qyl, knolw qyl Aqy sUrjmu`KI dw qly Aqy pSUAW dI crbI ijvyN ik murgI dI crbI Aq y bkrI dI crbI nUM ilipf dy bdlvyN sroqW vjoN cuixAw igAw[ m`CI dI Kurwk iv`c m`CI dy qyl nUM 0%, 25%, 50%, 75% Aq y 100% q`k ilipf dy bdlvyN sroqW nwl bdl ky iqAwr kIqI geI Kurwk pRXogSwlw hlwqW ADIn qzrby ADIn r`KIAW m`CIAW nUM 60 idnW q`k id`qI geI[ nqIijAW qoN ieh q`Q swhmxy Awey ik m`CI dI Kurwk iv`c m`CI dy qyl dI jgHw ilipf dy dUjy sroqW dI vrqoN krn nwl m`CI dy ivkws Aqy mwspySIAW dI pRogzImyt bxqr aupr koeI Kws pRBwv nhIN pYNdw[ m`CI dIAW mwspySIAW dy ku`l ilipf iv`c Kurwk dI PYtI AYisf bxqr dw pRBwv vyKx nUM imilAw[ v`Ko-v`KrIAW aupcwrq KurwkW vwlI sI. kwrpIE m`CI dIAW mwspySIAW iv`c SFAs, MUFAs Aqy PUFAs dI Gxqw iv`c ArQpUrn iviBMnqw pweI geI[ swrIAW KurwkW iv`c sbzI dy qyl/pSAU W dI PYt dI pRqISqqw iv`c vwDw hox nwl n-3 HUFAs dy p`DrW iv`c igrwvt AweI (p≤0.05)[ iesy qrWH, KurwkW iv`c sbzI dy qyl/pSAU W dI PYt dI pRqISqqw iv`c vwDw hox nwl n-3/n-6 PYtI AYisf dy AnupwqW iv`c igrwvt AweI[ n-3 PUFAs dy p`Dr dy G`t hox kwrn PYtI AYisf pRoPweIl dy ilhwz nwl sbzI dy qylW dy mku wbly pSUAW dI crbI AsMquilq pweI geI[ hwlWik m`CI dy qyl dI 25-50% imkdwr dI QW sbzI dy qyl/pSUAW dI crbI dI vrqoN nwl Kurwk iv`c n-3/n-6, PUFA/SFA Aqy PUFA/MUFA dw Anupwq isPwirS kIqIAW h`dW iv`c irhw[ auprokq qQ` nUM m`dynzr r`KidAW hoieAW, m`CI dIAW mwspySIAW dI PYtI AYisf bxqr aupr 100% m`CI dy qyl dw pRBwv vyKx leI kIqy gey Agl y qzrby leI 50% knolw Awiel Kurwk Aq y 50% poltrI PYt Kurwk nUM cuixAw igAw[ qzrby dy nqIijAW qoN pqw c`ilAw ik ijhnW m`CIAW nUM swrw smW kYnolw qyl Aqy poltrI PYt id`qI geI auhnW m`CIAW dy mku wbly mMfIkrn qoN 20 idn pihlW kYnolw qyl Aq y poltrI PYt qoN m`CI dy qyl qy ADwirq Kurwk vwlIAW m`CIAW dIAW PUFA dIAW GxqwvW iv`c vwDw hoieAw[ mu`K Sbd: i sprIns kwrpIE, m`CI dy qyl dw bdl, sbzI dy qyl, pSUAW dI crbI, PYtI AYisf pRoPwiel, ivkws, poRgzImty bxqr _________________ _______________ mu`K slwhkwr dy hsqwKr ividAwrQI dy hsqwKr CONTENTS CHAPTER TITLE PAGE(S) I INTRODUCTION 1-7 II REVIEW OF LITERATURE 8-23 III ACCEPTED/ PUBLISHED/SUBMITTED RESEARCH 24-98 ARTICLES IV GENERAL DISCUSSION 99-113 V SUMMARY 114-118 VITA CHAPTER I INTRODUCTION Aquaculture involves the farming of aquatic organisms such as fish, crustaceans and aquatic plants. Today, it is the fastest growing sector of animal production worldwide. During the last 25 years, aquaculture production grew up to 8.6% annually (FAO 2014) and currently covers roughly half the global demand for fish and fish products for human consumption. Annually, almost 100 million tonnes of fish are captured, of which around 70 million tonnes are intended for direct human consumption. Therefore, the constantly increasing demand for fish and fish products for human consumption must be covered by aquaculture. In addition to good growing conditions, a prerequisite for productivity and economic sustainability in fish farming is a reliable supply of effective feeds. One important ingredient of commercial aquaculture feeds is fish oil. Annual world production of fish oil fluctuates widely, as does its price. Nowadays, fish oil production varies within the range 0.9–1 million tons per annum (of which about 80% is exclusively used in aquaculture), with a market price of around 1000– 2000 USD per ton (FAO 2012). Fish oil is considered an excellent lipid source, especially in terms of polyunsaturated (PUFA) and highly unsaturated (HUFA) fatty acids (Rice 2009). Fish oil composition reflects the process whereby HUFAs produced mostly by microalgae are magnified in the food chain and consumed by humans as fish. These substances are highly valued with regard to human nutrition. In addition, the content of fat-soluble vitamins, primarily A and D (Rice 2009), and also antioxidants (e.g. vitamin E, carotenoids) in fish oil is of importance. In comparison with marine species, freshwater fish generally contain less n- 3 HUFA. This is due to the composition of the food chain. In marine environments, large amounts of n-3 HUFA are naturally present in algae, which are the sole primary producers in the food chain. Predatory species, standing at the top of the food chain, gain access to n-3 HUFA through pelagic fish. These small, oily fish are also the raw material for production of fish meal and fish oil. Therefore, the carnivorous fish produced in fed aquaculture contain high levels of n-3 HUFA in lipids. Due to increased living standards worldwide, human beings are becoming more and more health conscious, preferring to consume nutritious food with added health benefits. Fish as a food provides both. Fishes are a rich source of polyunsaturated fatty acids (PUFAs), mainly the n-3 PUFAs, besides having high protein content (15-25%), which is required for maintenance and growth of human body. Fishes are also rich in vitamins A, D, E and K and minerals (calcium, phosphorus and iron). Compared to beef and chicken, fish meat is more digestive as it contains much less connective tissue (Calder 2004). The fish fat is considered, most accessible and concentrated source of PUFAs. Interest has been growing in fish and fish products as a source of PUFAs, mainly of the n-3 family (also known as Omega-3 fatty acids). These include α-linolenic acid (C18:3 n-3) and long chain Omega-3 fatty acids (LC- PUFAs) or highly unsaturated fatty acids (HUFAs) viz. eicosapentaenoic acid (EPA; C20:5n- 3) and docosahexaenoic acid (DHA; C22:6 n-3). The n-6 family (also known as Omega-6 fatty acids) includes mainly linoleic acid (LA; C18:2 n-6) and arachidonic acid (AA; C20:4 n- 6). PUFAs are known to be cardio protective (Sanderson et al 2002), anti-atherosclerotic (Givens et al 2006), anti-thrombotic (Calder 2004) and anti-arrythmatic (Givens et al 2006). Studies on non-human primates and human newborns indicate that DHA is essential for normal development of retina and brain, particularly in premature infants (Montano et al 2001). Another PUFA of n-3 series, EPA decreases the risk of cancer development at certain sites (Harris 2004). The AA is a precursor of biologically important products such as epoxides and iso-prostanes, an isomer of prostaglandins (Galli and Marangoni 1997). The human body is capable of producing all the fatty acids it needs, except for two: LA, an omega-6 fatty acid, and alpha-linolenic acid (ALA), an omega-3 fatty acid. These have to be consumed from the diet and are therefore termed ―essential fatty acids‖. Both of these fatty acids are needed for growth and repair of tissues, but can also be used to make other fatty acids (e.g. AA is formed from LA). However, as conversion to omega-3 fatty acids EPA and DHA is limited, it is recommended that sources of these are also included in the diet. The ALA and LA are found in plant and seed oils. The EPA and DHA are found in oily fish (e.g., salmon, mackerel, herring). The AA can be obtained from animal sources such as meat and egg yolk. Human conversion of ALA to EPA is only about 15% and ALA to DHA is only about 5%. For that reason we need direct sources of EPA & DHA. The richest source of these fatty acids is fish (Mraz 2011). Different fish species have different range of PUFAs qualitatively and quantitatively. The fatty fishes, like hilsa and pangas contain 10-19% lipid content (and thus have higher PUFA value), whereas non-fatty fishes such as carps contain as low as 1-3% lipid (and are thus low in PUFA content). Also, fishes vary in their ability to produce EPA and DHA endogenously. Fatty acid composition in general and EPA and DHA contents in particular vary from species to species of fish and by seasonal and geographical parameters (Bimbo 2007). Therefore, the taste and nutritional quality of fish meat varies with season (Grun et al 1999; Ackman et al 2002), region (Ackman et al 2002) and diet (Grigorakis et al 2002). Marine water and freshwater fishes vary in their ability to produce PUFAs because of varying habitat and food. The PUFA content is higher in marine than in freshwater fishes. Content of EPA and DHA is more in marine than fresh water fishes because low temperature enhances elongation, food of marine fishes has more content of EPA & DHA and in freshwater fishes elongation enzyme is present but its content is low (www.foodsforlife.org). Fish meat containing favourable n-6 to n-3 ratios of PUFAs are considered to be the most beneficial in terms of human health. Any imbalance in n-6 to n-3 ratio is a significant 2