1 REPLACEMENT OF FISH OIL WITH PALM OIL

1 REPLACEMENT OF FISH OIL WITH PALM OIL IN DIETS OF NILE TILAPIA (OREOCHROMIS NILOTICUS) AFFECTS FATTY ACID COMPOSITION AND GENES INVOLVED IN LIPID SYNTHESIS AND TRANSPORT PRESENTED AT SECOND COLLOQUIUM SERIES (2018/2019), FACULTY OF NATURAL RESOURCES AND ENVIRONMENT CHRISTIAN LARBI AYISI (Ph. D) 2/25/2021

2 CONTENT INTRODUCTION AND OBJECTIVES MATERIALS AND METHODS RESULTS AND DISCUSSION -III CONCLUSION & RECOMMENDATIONS 2/25/2021

ABBREVIATION Enzymes, genes and transcriptional factor 3 Abbreviation Function Peroxisome proliferator-activated receptor PPAR Transcription factor Retinoid X receptor gamma variant a RXR Transcription factor Sterol regulatory element binding protein SREBP Transcription factor Malic enzyme ME Production of NADPH Fatty acid binding protein FABP Fatty acid transport Fatty acid translocase/cluster of differentiation 36 FAT/CD 36 Uptake of Fatty acid Lipoprotein lipase LPL Uptake of Fatty acid Microsomal triacylglycerol transfer protein MTP Fatty acid transport Glucose 6 -phosphate dehydrogenase G 6 PD Fatty acid transport 6 -phosphogluconate dehydrogenase 6 GPD Fatty acid transport Acetyl-Co. A Oxidase ACO β-oxidation Acyl-Co. A dehydrogenase, very long chain ACDHVL β-oxidation Acyl Co. A De. Hydrogenase ACDH β-oxidation Acetyl-Co. A acyltransferase 2 (thiolase) ACAT 2 β-oxidation Carnitine Palmitoyltransferase a CPT 1 β-oxidation I CPT 2 β-oxidation Mitochondrial carnitine palmitoyltransferase 2/25/2021 alpha 1 a Elongases of very long-chain fatty acids ELOVL Elongation

4 INTRODUCTION Achieving Food Security: AGlobal Expected population by 2050 Concern 2/25/2021

5 REALITY v More than 800 million people are suffering from chronic malnourishment v Majority of the World’s hungry people live in developing countries v Asia is the continent with the most hungry people v Sub-Saharan Africa is the region with the highest prevalence (1/4 persons is undernourished) v World leaders are renewing their commitment to make nutritious diets available to all 2/25/2021

WHAT IS THE WAY FORWARD 6 2/25/2021

7 122. 9 151. 8 2/25/2021

8 AQUACULTURE OUR HOPE FOR NUTRITION? 2/25/2021

Fish production (Capture fisheries and Aquaculture): 1991 -2024 9 Inter annual variations never exceeded 1% Even exceptionally stable 2/25/2021

Share of global aquaculture in fish production, 1997 and forecast for 2020 Fish production 1997 Increase in fish production, 2020 Source: Rosegrant 2003

CHALLENGES OF AQUACULTURE POORLY COORDINATED EXTENSION SERVICES POST HARVEST LOSSES POOR FINGERLINGS DISEASES HIGH COST OF FEED/FEEDING REDUCTION IN PRODUCTION AND PROFITS

12 HIGH COST OF FEED Feed accounts for about 60 -70% of total aquaculture production Fish meal and fish oil are major ingredients in fish diets Fish meal and fish oil are highly localized Fish meal and fish oil are expensive 2/25/2021

FISH OIL 13 l. Rich in n-3 LC-PUFA l. Monounsaturated fatty acids (MUFA) l. Higher prices/ Expensive l. Declining/ Unsustainable l. Unable to meet demands of aquaculture l. Highly localized hence difficult to obtain in some regions 2/25/2021

FISH OIL PRODUCTION AND UTILIZATION 14 Fish oil production About 73% of FO is used in aquaculture. 2/25/2021

Any alternatives? 15 VEGETABLE OILS: v Palm oil v Sunflower oil v Peanut Oil v Corn Oil v Canola oil ANIMAL OILS v Pork v Poultry Fat v Tallow 2/25/2021

PALM OIL 16 ü Relatively Cheaper üAbundant/ sustainable üRelatively low organic contaminants üRich source of vitamin E üResist feed rancidity üExcessive levels of n-6 PUFA üLack of n-3 PUFA üLow levels of EPA and DHA 2/25/2021

Production of palm oil from 2012/2013 to 2017/2018 17 by type (Million metric tons) PROJECTION 2/25/2021

RECENT STUDIES ON REPLACEMENT OF FO WITH 18 PO üNg et al. 2000 üOchang et al 2007 üKomilus et al. 2008 üBabalola and Apata, 2012 üHan et al. 2012 üSingh et al 2012 üHan et al 2015 üHuang et al. 2016 üAnvo et al. 2017 Bagrid catfish Nile tilapia Red sea bream Catfish Japanese sea bass Cirrhinus mrigala Japanese flounder Chu’s Croaker Catfish 2/25/2021

19 Consequence of replacing FO with PO in fish diets ü Excessive lipid deposition in tissues üChanges whole body/fillet Fatty acid composition üCost effective 2/25/2021

What next? 20 ü Does different oil sources affect nutritional composition? ü Why does replacement of FO with PO cause lipid deposition in the liver? üDoes different oil sources affect lipid metabolism pathways? 2/25/2021

OBJECTIVES 21 The objective is to evaluate how replacement of FO with PO affects; v. Growth, feed utilization and serum metabolites v. Fatty acid composition, nutritional value and m. RNA expression of genes involved in lipid metabolism vm. RNA expression of genes and signaling factors related to fatty acid transport 2/25/2021

22 MATERIALS AND METHODS 2/25/2021

23 100% FO, 0% PO EXPERIMENTAL DIETS 75% FO, 25% PO 50% FO, 50% PO 25% FO, 75% PO 0% FO, 100% PO 2/25/2021

Formulation, proximate composition (g kg-1 ) of experimental diets Ingredients (g kg-1 diet) 0% PO 25% PO 50% PO 75% PO 100% PO Fish meal* 60 60 60 Soybean meal* 300 300 300 Wheat meal* 225 225 225 Rapeseed meal* 300 300 300 Fish oil* Palm oil* 60 0 45 15 30 30 15 45 0 60 Soybean phospholipid* 25 25 25 Mineral mix 5. 5 5. 5 Vitamin mix 4 4 4 Ca(H 2 PO 4) 15 15 15 Choline Chloride 5 5 5 Inositol 0. 5 0. 5 Moisture 103 105. 3 108. 2 107 105 Crude protein (dry 331. 8 mass) Lipid (dry mass) 98. 2 331. 5 330. 9 332. 3 331. 7 98. 1 98. 7 98. 8 98. 5 Ash (dry mass) 56 55. 3 54 55 2/25/2021 24 52. 5 IN ACCORDANCE TO NRC, 2011

Main fatty acid composition of experimental diets (g/kg) Fatty acid composition (g kg 0% PO -1) 25% PO 50% PO 75% PO 100% PO 12: 0 1. 5 1. 4 1. 2 1. 1 14: 0 55. 4 45. 2 32. 0 22. 5 12. 7 16: 0 238. 8 259. 0 270. 0 280. 6 291. 9 18: 0 53. 1 57. 6 54. 3 53. 0 51. 5 Σ SFA’s 348. 8 36. 32 357. 5 357. 3 357. 2 16: 1 n-7 60. 2 52. 3 31. 5 20. 6 8. 9 18: 1 n-9 235. 6 254. 2 284. 0 311. 6 337. 7 Σ MUFAs 295. 8 306. 5 315. 5 332. 2 346. 6 18: 2 n-6 206. 5 215. 7 230. 7 236. 2 243. 3 20: 4 n-6 5. 6 4. 7 4. 1 2. 5 2 Σ n-6 212. 1 220. 4 234. 8 238. 7 245. 3 18: 3 n-3 55. 2 39. 6 39. 8 37. 9 36. 3 18: 4 n-3 3. 2 3. 0 2. 9 20: 5 n-3 42. 5 31. 9 22. 0 14. 1 6. 2 22: 6 n-3 56. 6 41. 5 28. 5 16. 9 5. 8 Σ n-3 157. 5 116. 3 93. 5 71. 9 51. 2 DHA/EPA 13. 3 13. 0 12. 9 11. 9 9. 3 Σ PUFAs 349. 6 336. 7 328. 3 310. 6 296. 5 ΣSFA/Σ PUFA 8. 7 10. 8 11. 5 12. 0 Σn-3/Σn-6 7. 4 5. 2 3. 9 3. 0 2/25/2021

MATERIALS AND METHODS 26 Transported to rearing base. Acclimatized for two week. Fed feed from Jin Yuan Trade twice a day for two weeks Nile tilapia (9. 34 g) collected from Tilapia germplasm station 2/25/2021

MATERIALS AND METHODS CONT. 27 ü 15 fiber tanks (150× 60× 40 cm) üwater maintained at 210 litres üStocked at 40 fish per tank ü Twice daily üApparent satiation FEEDING: ü 8: 00 am & 4: 00 pm ü 8 weeks 2/25/2021

28 Sampling and Analysis Sampling Size: Five (5) fish per tank Feed withheld 24 hours before sampling Length and weight measured in cm and g respectively 2/25/2021

29 Biochemical analyzer (Mindray Chemistry Analyzer BS-200, Shenzhen China). Blood was collected from caudal veins, centrifuged and serum removed and stored at -20°C for subsequent analysis 2/25/2021

Estimation of Growth and Feed Utilization 30 Feed Intake (g) = total feed eaten (g) during culture period Weight Gain (WG) = Final weight (g) – Initial weight (g) Condition factor (C) = [Body weight/ (Total length) 3] × 100% 2/25/2021

31 NUTRITIONAL QULAITY v Polyene Index (PI)= C 20: 5+C 22: 6/ C 16: 0 (Lubis and Buckel, 1990) v Thrombogenic Index (TI)= (C 14: 0+C 16: 0+C 18: 0)/ [(0. 5* Sum MUFA)+(0. 5*n-6 PUFA)+(3*n-3 PUFA)+ (n-3: n-6) (Ulbricht and Southgate, 1991) v Peroxidase Index (Pe. I)= 0. 025*(% monoenoic)+ 1*(% dienoics) + 2*(trienoics) + 4*(% tetraenoics) + 6 (% pentanoics) + 8 (% hexaenoics) (Hulbert et al. , 2007) 2/25/2021

32 LABORATORY ANALYSIS Fatty acid analysis (feed, liver and muscle) • Lipid extracted using Folch et al 1957 method. • Fatty acid methyl esters separated using Gas Chromatography Machine (GC 7890 A, USA. • Fatty acids identified by comparing peak times of samples compared to that of standards Enzyme activities Analyzed using test kits from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Procedures as described by kit manufacturer was followed. 2/25/2021

GENE EXPRESSION / PROFILING 33 üReal-time PCR: Mini Option Real-time PCR machine (Bio-Rad). ü The 20 -µl reaction contained: l 1 -µl c. DNA sample l 10 µl SYBR green I Master Mix (Ta. Ka. Ra) l 0. 5µl of each primer l 8 µl deionized water. üPCR amplification: l l l Triplicate wells 3 min at 95°C 45 cycles consisting of 10 s at 95 °C, 15 s at 63°C 25 s at 72°C. 2/25/2021

34 ü A melting curve analysis was performed to confirm that a single PCR product had been amplified. üAt the end of the reaction, the fluorescent data were converted into Ct values. üEach transcript level was normalized to β-actin using the 2 - Δ Δ CT method (Livak, & Schmittgen, 2001). ü All primers designed using primer 5. 2/25/2021

Genes 35 Table 3: Primer sequences used for real-time quantitative PCR (q. RT-PCR). Forward (5′– 3′) Lipid synthesis/breakdown TTTGAGATGTGCTCACAGCTGC FASN ACC SCD 1 ACYL CPTIa CPTIb Lipid transport PPAR-α PPAR-β LPL FABP 3 ME MTP LXR ELOVL-5 6 GPD G 6 PD β –ACTIN AGA ATTAAACACTAAAGAAGAAGA GCTT GACATTTTTGCCTTTTTAGA GCACTGTTCAGATGGTTTATGT TTATGACCT GTACGTCTGTTCGCTCCTGCAC CAA AAGCCACTTCAAGGCATAGGA ATC TACGGTGTTTACGAAGCCCT ACCTGGGCTAATGAACGTGA ATTGCCGGAGACCTTACCAA TCTCTCATTCTGACGCTCCC CGATGGGGATTGCTGAACTG CCTCTACGCATGTGGTTTCG ACTAAGTAGCGTCCACTCGG GGCTTCCTCCTCCGTCTAAA GAGAGTCGTGGCCAGTAAGA TGCTCCTGTTTCTCCG ATCGTGGGGCGCCCCAGGCATC AGG Reverse (5′– 3′) Gen. Bank accession no. TCTGCAGCTGTGAGCACATCTCAAA GU 433188. 1 AAGCTCTTCTTCTTTAGTGTTTAAT XM_003442879. 3 TCCTAATAAGAGTATA AGGTCATAAACCATCTGAACAGTGC AJ 55697 XM_005470605. 2 TTGGTGCAGGAGCGAACAGACGTAC DQ 011056. 1 GATTCCTATGCCCTTGAAGTGGCTT NM_001171855. 1 AGGAAGGTGTCATCTGGGTG CTGTAGTAGAGGGTGGAGCG TGGTCTCTGGATGCCGATAC CGCCCAGCTCTTTCATGTAC ATCTGTGGGTGCTCATGTGA AGGATCAGCTGCTTCACCTT GGGCAGAAAGGAGTCAGTGA GTGCAAAGGTTGGTGGGTAG ACCAGCCGAACTCTTTAGCT CATCCCAGCGTTCATTCCTGATGCCTGGGGCCCACGAT KF 871430. 1 KF 751705. 1 GU 433189. 1 XM_003444047. 4 XM_003453476. 4 KU 954530. 1 FJ 502320 NM_001279460. 1 JX 992745 JX 992744 2/25/2021 EU 887951. 1

36 STATISTICAL ANAYSIS üData was analyzed using One-Way ANOVA (Non-Parametric) üCorrelation between tissue fatty acids and gene expression were analyzed üTukey’s multiple tests was used to compare means of all treatments. üSignificant differences was tested at P<0. 05. üGraph Pad Prism (V. 5. 03) was used to perform all analysis üData presented as mean ± standard error of the mean (SEM). 2/25/2021

RESULTS AND DISCUSSION I 37 Effects on growth, feed utilization and serum biochemical composition Table 4: Growth performance and feed utilization of Oreochromis niloticus fed experimental diets Dietary Palm oil (PO) replacement level (%) Parameters Final weight (g) WG PO 0 PO 25 PO 50 PO 75 PO 100 P value 66. 34± 1. 03 ab 62. 48± 0. 91 a 69. 85± 2. 19 b 64. 42± 1. 15 ab 65. 05± 1. 11 ab 0. 032 56. 98± 0. 96 ab 53. 18± 1. 26 a 60. 54± 2. 14 b 55. 04± 0. 71 ab 55. 63± 1. 17 ab 0. 030 SGR (%/d) 3. 52± 0. 00 3. 40± 0. 04 3. 55± 0. 07 3. 44± 0. 01 3. 45± 0. 03 0. 126 FI (g) FCR 76. 74± 0. 21 1. 34± 0. 02 76. 27± 0. 26 1. 43± 0. 03 77. 64± 0. 26 76. 69± 0. 39 1. 39± 0. 02 77. 19± 0. 35 1. 38± 0. 03 0. 076 0. 070 C 2. 14 ± 0. 06 1. 99 ± 0. 06 2. 16 ± 0. 06 2. 07 ± 0. 06 2. 02 ± 0. 04 0. 278 HSI 1. 78± 0. 11 1. 91± 0. 10 2. 02± 0. 11 2. 07± 0. 11 2. 18± 0. 125 1. 28± 0. 03 l. Tilapia like some other fresh water fish have greater requirement for n-6 FA l. EFA are met when a significant proportion of FO is replaced by other lipids (VO) 2/25/2021 l. FI and FCR: Not significantly different. Signify equal acceptability and ability to digest and convert feed to body weight.

Serum biochemical indices Parameters Dietary replacement level (%) PO 0 25 50 75 100 P- value TC (mmol L-1)1 3. 51± 0. 16 3. 52± 0. 07 3. 75± 0. 24 3. 89± 0. 30 3. 99± 0. 05 0. 335 TP (mmol L-1)2 30. 72± 1. 05 a 35. 32± 1. 19 ab 36. 81± 1. 85 b 36. 87± 0. 81 b 35. 10± 1. 05 ab 0. 014 TG (mmol L-1)3 3. 26± 0. 23 a 4. 03± 0. 03 ab 4. 76± 0. 17 bc 5. 16± 0. 43 bc 5. 57± 0. 40 c 0. 000 HDL-C (mmol L-1)4 2. 32± 0. 33 2. 30± 0. 27 2. 40± 0. 13 2. 19± 0. 15 1. 60± 0. 07 0. 253 LDL-C (mmol L- 0. 27± 0. 02 a 1 )5 0. 30± 0. 2 ab 0. 32± 0. 02 ab 0. 42± 0. 04 b 0. 43± 0. 01 b 0. 007 AST (UL-1)7 17. 50± 1. 83 a 20. 86± 1. 69 ab 24. 58± 1. 86 ab 23. 72± 1. 46 ab 39. 02± 1. 05 b 0. 029 ALP (UL-1)8 17. 21± 1. 23 a 30. 09± 1. 78 b 32. 99± 1. 07 b 46. 85± 1. 90 c 0. 0001 38 33. 30± 19 b ALP: increased with increasing PO. l. This imply a possible damage of liver parenchyma cells. AST: 100% PO significantly higher than 0% PO. l. This could be due to a possible liver injury or damage. TG: 50% PO, 75% PO and 100% PO significantly higher than 0% PO. l. Vegetable oils are known to enhance secretion of TG 2/25/2021

II Fatty Acid Composition, Nutritional value and m. RNA Expression of 39 Genes Involved in Lipid Metabolism Fatty acid(s) PO 0 Dietary PO replacement level (%) PO 25 PO 50 PO 75 PO 100 P-value 7. 48± 0. 86 7. 63± 0. 43 8. 46± 0. 59 8. 61± 0. 78 9. 06± 0. 41 0. 1065 96. 4± 0. 00 a 106. 0± 1. 4 b 120. 4± 3. 9 b 141. 3± 1. 7 c 145. 6± 6. 7 c 0. 0001 18: 3(n-3) LNA 9. 1± 0. 2 9. 8± 0. 1 9. 6± 1. 5 10. 0± 4. 9 11. 5± 0. 00 0. 8624 20: 4(n-3) 9. 6± 0. 01 b 9. 8± 0. 02 7. 7± 1. 5 b 6. 8± 0. 3 9. 1± 0. 1 b 7. 2± 0. 1 3. 8± 0. 00 a 10. 0± 2. 1 2. 9± 0. 1 a 8. 7± 0. 1 0. 0017 0. 4513 20: 5(n-3) (EPA) 22: 6(n-3) (DHA) 10. 6± 0. 2 b 5. 8± 1. 5 ab 2. 0± 0. 3 a 2. 7± 0. 3 a 1. 3± 0. 00 a 0. 0014 63. 5± 1. 8 c 58. 5± 3. 1 c 43. 3± 0. 9 b 14. 7± 1. 9 a 8. 5± 0. 02 a 0. 0001 ΣSFA’s Σ MUFAs Total PUFAs Σ n-3 Σ n-6 371. 8± 4. 6 a 389. 8± 8. 5 a 232. 7± 2. 0 c 101. 6± 2. 2 e 131. 0± 0. 2 ab 7. 7± 0. 4 c 377. 6± 8. 3 b 397. 9± 4. 7 b 214. 8± 4. 6 bc 88. 1± 8. 9 d 126. 6± 2. 1 a 6. 9± 0. 2 c 382. 5± 1. 9 c 421. 4± 7. 7 c 208. 6± 4. 3 b 68. 8± 3. 3 c 139. 8± 3. 6 abc 4. 9± 0. 1 b 395. 7± 3. 3 d 424. 2± 4. 3 d 191. 7± 43. 1 ab 36. 5± 1. 8 b 155. 2± 1. 3 bc 2. 4± 0. 1 a 397. 0± 7. 2 e 435. 5± 3. 2 e 185. 0± 7. 0 a 27. 7± 6. 2 a 157. 3± 6. 8 c 1. 8± 0. 00 a 0. 0056 0. 0001 0. 0007 0. 0001 0. 0033 0. 0058 Lipid 18: 2(n-6) 20: 4(n-6) n-3: n-6 LA ARA l. Fatty acids in mirrored that of their corresponding feeds ln-3/n-6 decreased as PO increased. This is because n-3 FA had been spared by 2/25/2021 MUFA and to some extent SFA l. This was caused by the increase in 18: 2 n-6 and decrease LC-n-3 PUFA

40 l. DHA in liver greater than DHA in feed. This imply selective retention (Mozanzadeh et al. 2016) l Imply ability to synthesize EPA and DHA from short chain FA such as ALA (Han et al. 2013) l Nile tilapia was able to convert LA to PUFA such as 20: 4 n-6 (ARA). 2/25/2021

41 Nutritional Quality (Muscle) 2/25/2021

42 l. Lower PI represent decomposed PUFA l. This could imply increase in thiobabituric acid index l. TI shows the tendency to form clots in the blood vessels l. Relationship between pro-Thrombogenic and anti-throbogenic fatty acids l. Diminishing values are better. 2/25/2021

Muscle fatty acids 43 Fatty acid(s) Muscle lipid ΣSFA’s Σ MUFAs 16: 3(n-3) 18: 3(n-3) 20: 4(n-6) (ARA) 0% PO 94. 8± 1. 2 352. 3± 1. 3 a 438. 4± 1. 2 7. 7± 0. 1 c 5. 0± 0. 4 a Dietary PO replacement level (%) 25% PO 50% PO 75% PO 98. 2± 3. 8 95. 4± 0. 5 98. 9± 7. 4 a b 356. 0± 3. 4 400. 6± 3. 4 406. 8± 3. 5 b 432. 9± 0. 5 436. 2± 8. 9 450. 2± 2. 5 6. 5± 0. 7 bc 5. 5± 0. 1 b 5. 2± 0. 3 b 8. 1± 0. 00 ab 10. 6± 1. 1 bc 12. 2±± 0. 3 c 100% PO 91. 5± 0. 8 407. 5± 1. 9 b 441. 8± 4. 0 3. 2± 0. 3 a 11. 6± 0. 9 c p-value 0. 6681 0. 0345 0. 6738 0. 0012 0. 0041 5. 4± 0. 1 ab 5. 2± 0. 3 a 5. 8± 0. 2 ab 6. 5± 0. 2 b 0. 0229 8. 4± 0. 7 bc 7. 7± 1. 0 abc 5. 6± 0. 5 ab 4. 7± 0. 2 a 0. 0018 43. 2± 0. 3 c 40. 9± 2. 6 c 24. 6± 1. 8 b 15. 7± 0. 6 a 0. 0006 71. 7± 1. 3 c 70. 6± 3. 6 c 53. 7± 0. 8 b 41. 8± 0. 7 a 0. 0002 20: 5(n-3) (EPA) 9. 7± 0. 3 c 22: 6(n-3) (DHA) 48. 0± 0. 7 c 75. 9± 0. 3 c Σ n-3 18: 2(n-6) LA 105. 9± 4. 3 ab 124. 8± 6. 1 b 103. 6± 1. 9 a 103. 0± 2. 1 a 109. 9± 4. 3 ab 0. 0021 20: 2(n-6) 20: 4(n-6) 16. 2± 1. 3 d 8. 4± 0. 2 c 12± 0. 7 c 7. 1± 0. 6 b 6. 8± 0. 1 b 5. 7± 1. 0 ab 5. 0± 0. 4 ab 4. 4± 0. 1 a 2. 6± 0. 00 a 3. 5± 0. 1 a 0. 0010 0. 0027 Σ n-6 130. 4± 3. 8 b 144. 6± 7. 0 c 116. 2± 1. 5 ab 112. 5± 2. 4 ab 116. 1± 4. 3 a 0. 0401 Total PUFAs 206. 3± 3. 8 cd 216. 3± 5. 8 d 186. 9± 4. 3 bc 166. 2± 5. 0 ab 158. 0± 5. 0 a 0. 0001 n-3: n-6 EPA+DHA 5. 8± 0. 1 cd 57. 7 c 4. 9± 0. 3 bc 51. 6 c 6. 0± 0. 2 d 48. 6 c 4. 7± 0. 1 b 30. 2 b 3. 6± 0. 00 a 0. 0366 20. 4 a 0. 0009 2/25/2021

Gene expression profiles of lipid synthesis/metabolism related genes in liver 44 Acetyl-Co. A carboxylase (ACC) ATP citrate lyase (Acyl) l. ACC and FASN are regulated coordinately (Toussant et al. , 1981) l. Higher expression of m. RNA of FAS and ACC coincided with the 2/25/2021

45 Fatty acid synthase Stearoyl-Co. A desaturase FAS: l. This is because PUFA inhibited FAS expression (Leng et al 2012) 2/25/2021

46 l. Higher SFAs with lower PUFAs upregulated SCD 1 l. Supports earlier report by Hsieh et al. 2007 in tilapia l. Might have caused the increase in liver lipid content, even though not significantly different l. This is because up-regulation of SCD 1 does not inhibit lipid synthesis. 2/25/2021

Carnitine palmitoyltransferase 47 l. CPT 1 a and CPT 1 b decreased as PO decreased l. Reduction in the n-3 LC-PUFA as PO increased might be the reason because CPT 1 has higher affinity to n-3 LC PUFA l. PUFA affects CPT 1 activity through the changes in the mitochondrial membrane composition (Jackson et al. 2000). 2/25/2021

48 CPT 1: Correlates with C 18 PUFA specifically 18: 2 n-3. l. This imply increasing PO which reduces C 18 leads to storage of LC-PUFA instead of beta oxidation l. This is because n-3 LC-PUFA possibly increased mitochondrial fatty acid oxidation hence stimulating CPT 1 activity (Madsen et al. 1999) 2/25/2021

III 49 Substitution of fish oil with palm oil affects enzyme activities and m. RNA expression of lipid transport related genes in Oreochromis niloticus Hepatic enzyme activity of lipid transport genes Gene (U/L) 0% PO PPAR-α PPAR-β LPL FABP-3 ME MTP LXR ELOVL-5 6 GPD G 6 PD 9. 32± 1. 26 a 8. 13± 0. 832 a 29. 47± 1. 38 a 10. 70± 0. 13 a 82. 69± 9. 83 192. 81± 6. 33 53. 77± 3. 89 b 244. 81± 8. 61 b 46. 79± 5. 69 25. 60± 2. 45 Dietary palm oil inclusion level (%) 25% PO 50% PO 75% PO 11. 85± 1. 13 ab 13. 87± 1. 88 b 39. 56± 5. 24 ab 11. 40± 0. 55 a 80. 00± 7. 59 195. 01± 2. 81 32. 63± 2. 34 a 166. 8± 8. 40 a 55. 94± 5. 15 29. 19± 2. 72 17. 71± 2. 52 bc 10. 14± 0. 95 ab 61. 17± 5. 87 bc 16. 94± 2. 67 ab 74. 50± 8. 53 199. 51± 9. 71 34. 75± 3. 49 a 188. 6± 3. 57 a 59. 68± 3. 04 31. 02± 2. 23 18. 08± 1. 63 bc 8. 34± 0. 71 a 66. 47± 9. 59 c 20. 81± 2. 39 b 94. 14± 10. 94 210. 01± 6. 88 33. 26± 2. 36 a 163. 5± 5. 30 a 54. 47± 5. 20 30. 53± 2. 68 100% PO P-value 20. 36± 2. 06 c 9. 16± 0. 47 a 67. 43± 6. 92 c 20. 54± 3. 29 b 79. 93± 4. 32 216. 5± 7. 766 27. 97± 1. 02 a 161. 5± 7. 80 a 60. 93± 2. 58 27. 69± 2. 70 0. 0008 0. 0062 0. 0005 0. 0069 0. 5830 0. 7765 0. 0368 0. 0001 0. 2271 0. 5734 2/25/2021

50 Liver X receptor Lipoprotein lipase 2/25/2021

51 l. LPL: m. RNA expression of 100% PO significantly higher than all other groups. Enzyme activities of 50% PO, 75% PO and 100% PO significantly higher than 0% PO u 1. Half life of LPL is very short hence seems to be controlled by its enzyme activities as in the case of 100% PO. u. Regulation of LPL seems to take occur at the transcriptional level in response to changes in liver fat content (Tian et al. 2013). This is why 50% PO and 75% PO had enzyme activities higher than 0% PO but not their m. RNA expression. l. Higher expression of LPL in 100% PO indicates there was dire need for lipid in this group to be stored or oxidized to maintain balance. l. LXR: Reduction in LXR could be due to higher levels of n-6 and lower levels of n-3 PUFA in diets and liver l. It signifies PO antagonize or suppress LXR expression 2/25/2021

52 Glucose 6 -phosphate dehydrogenase 6 -phosphogluconate dehydrogenase 2/25/2021

53 Malic enzyme Peroxisome proliferator-activated receptor 2/25/2021

54 2/25/2021

l. PO up regulated G 6 PD and 6 GPD even though 6 GDP not significantly different. 55 l. G 6 PD and 6 GPD release NADPH compounds that aid in FA synthesis l. Increase in G 6 PD and 6 GPD and their correlation with enzyme activities indicate they play essential role in lipid metabolism when FO is replaced with PO. l. It also suggest replacing FO with PO does not deplete supply of NADPH l It shows PO can promote lipid synthesis and metabolism l. PO up regulated PPAR-α and PPAR -β even though PPAR -β not significantly different. l. This is because higher levels of n-6 fatty acids attracts activity or expression of PPARs l. It suggest increasing PO in diets increases β-oxidation because PPAR-α is an indicator of β-oxidation (Zuo et al 2013, Zheng et al. 2013) 2/25/2021

Principal component analysis

Principal component analysis • The upper hemisphere was occupied by only LXR whilst the lower hemisphere was occupied by ME, LPL, 6 GPD, G 6 PD, FBP-3, PPAR-α, PPAR-β and MTP. • With respect to fatty acids, 20: 4 n-3, 20: 5 n-3, 22: 6 n-3, Σ PUFA, Σ n-3 as well as Σ n-3: Σ n-6 ratio occupied the upper hemisphere whilst 18: 3 n-6, 20: 4 n-6, 18: 2 n-6, Σ n 6, Σ SFA, Σ MUFA and Σ SFA/ Σ MUFA occupied the lower hemisphere.

Correlation analysis between acids and lipid m. RNA expression of related genes fatty BROWN COLOURS CORRELATE NEGATIVELY WHILST BLUE COLOURS CORRELATE POSITIELY. BOXED SYMBOLS CORRELATE SIGNIFICANTLY, THE BIGGER THE SYMBOLS, THE STRONGER THE CORRELATION

CONCLUSION 59 l. Feeding Nile tilapia with palm diets does not compromise growth performance. l. Substituting FO with PO affects fatty acid composition in liver and muscle v 20: 4 n-6, 20: 5 n-3, 22: 6 n-3 as well as n-3 PUFA reduced in both liver and muscle l. Substituting FO with PO alters the lipid synthesis metabolism and transport pathways v PPAR-α, Malic enzyme, LPL, LXR and G 6 PD expression are regulated by Fatty acids v FAS, SCD 1, ACYL, ACC and CPT I are influenced by substituting FO with PO 2/25/2021

RECOMMENDATIONS 60 v Fish oil should be replaced with Palm oil at 50% v Local ingredients should be investigated to know their efficacy v Genes that are closely related to long-chain fatty acids should be knocked-out in future experiments 2/25/2021

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