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Towards Development of a Formulated Diet for Blue Swimmer Crab, Portunus Pelagicus with Emphasis on Lipid Nutrition

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Towards Development of a Formulated Diet for Blue Swimmer Crab, Portunus Pelagicus with Emphasis on Lipid Nutrition ResearchOnline@JCU This file is part of the following reference: Mat Noordin, Noordiyana (2011) Towards development of a formulated diet for blue swimmer crab, Portunus pelagicus with emphasis on lipid nutrition. PhD thesis, James Cook University. Access to this file is available from: http://researchonline.jcu.edu.au/39433/ The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] and quote http://researchonline.jcu.edu.au/39433/ Towards development of a formulated diet for blue swimmer crab, Portunus pelagicus with emphasis on lipid nutrition Thesis submitted by Noordiyana Mat Noordin in June 2011 for the Degree of Doctor of Philosophy in the School of Marine & Tropical Biology James Cook University DECLARATION ON ETHICS The research presented and reported in this thesis was conducted within the guidelines for research ethics outlined in the National Statement on Ethics Conduct in Research Involving Human (1999) the Joint NHMRC/AVCCStatement and Guidelines on Research Practice (1997), the James Cook University Policy on Experimentation Ethics. Standard Practices and Guidelines (2001) and the James Cook University Statement and Guidelines on Research Practice (2001). The proposed research methodology received clearance from the James Cook University Experimentation Ethics Review Committee: Approval number: A1285 Signature: Name: Noordiyana Mat Noordin Date: June 23rd, 2011 i Acknowledgements Alhamdullilah, Once, a professor told me that doing PhD is a process that involves mental, physical and emotional torture. I laughed. Few years later, here I am agreeing in every single word that he said. I could have not done this without help and support of people around me and I will be forever indebted to them. First and foremost, I would like to thank my supervisors, Dr Chaoshu Zeng and Professor Paul Southgate, for being a torch shining my path and guiding me all along. I could not ask for better supervisors. To Su Reilly of JCU histological lab and Ian Brooks of Department of Primary Industries and Fisheries, Queensland, thank you for teaching and helping me with my samples analysis. To Greg, Simon and John of MARFU, thank you for helping me with my lab set up. I also could not complete my PhD without the helped of my research mate especially Nick Romano, Jerome Genodepa and Wu Xugan, who at the end of my study has become more like my brothers than just my friends. To Maya, Malwine, Kiki, Jonathan, Michael, Matt and Laura, thank you for offering hands and motivation when I needed. Not to forget, to people who paint my life with colors in Australia; Azwita and family, Miin, Anne, Mandy, Alan and whole bunch of crazy people in Rotary International House 2006-2009, you guys are awesome. Last but not least, to Nazipah, Mat Noordin, Azwa, Nirwandy, Hezly, Lin, Naem, Wafri, Darwish, Fatihah, Fara and Amir, no word will be enough to show my appreciation to all of you. You are my everything and thank you for always believing in me. ii Abstract Market demands on the blue swimmer crab, Portunus pelagicus, have increased substantially worldwide over past decades and have expanded from traditional hard-shell crabs to soft-shell crabs as well as pasteurized crabmeat. Increasing fisheries pressure has led to declining wild population and aquaculture has been eyed as the alternative to supply the market. As an emerging aquaculture species, comprehensive and quantitative understanding of nutritional requirements of P. pelagicus is necessary to the development of formulated diets for the species to support sustainable growth of the industry. As a crucial nutrient, lipid provides cultured crustaceans with concentrated energy, components of cell membrane and as precursors for various hormones. A series of experiments were hence conducted to evaluate the necessity as well as optimal levels of various lipid components for P. pelagicus early juveniles. Semi-purified diets were formulated to contain different levels (% diet dry weight) of a particular lipid component and the diets were fed to newly molted first stage crabs (C1) till the crabs reached C3 or C4 stage with survival, development and growth of crabs subjected to different dietary conditions closely monitored. To prevent cannibalism, all experimental crabs were kept individually for all experiments. A starvation and re-feeding experiment was first conducted to investigate their effects on survival and development of newly settled C1 crabs as well as their fatty acid profiles. Meanwhile, hepatopancreas histology was undertaken to observe morphological changes iii induced. Three treatments: 1) continuous starvation (S); 2) starved for 7 days and then re-fed (S-F); and 3) continuous feeding (F), were set up. In S treatment, crab survival declined rapidly (from 96.6% to 47.9%) between day 7 to 10, leading to the termination of the treatment on day 10. The PNR50 (point-of-no return) for the C1 crabs is therefore between day 7 to 10. There was no significant differences on crab survival between S-F and F treatments, however, newly molted C2 and C3 of S-F treatment had significant lower dry weights (p<0.05). Interestingly, despite their significant lower dry weight, S-F crabs had a significant shorter mean intermolt period from C2 to C3 than that of F crabs (p<0.05). This suggests that following prolonged starvation, re-fed crabs prioritized development over growth. Fatty acid profiles of starved crabs showed a trend of decreased PUFA but increased HUFA (as % total fatty acids), suggesting high retention of HUFA. S-F crabs managed to recover their fatty acid profiles after being re-fed and were similar to F crabs as newly molted C2. Histology observation showed a continuous shrinking of hepatopancreas cells during starvation, however, as newly molted C2 and C3, no obvious differences was noticed between the re-fed and continuously fed crabs. A series of 8 feeding experiments were then subsequently conducted to assess dietary cholesterol and phospholipid (PL) requirements, cholesterol and PL interactions, neutral lipids (triglyceride) requirements, fish oil to soybean oil ratio, arachidonic acid (ARA) requirements and polar lipids (PL) to neutral lipids (triglyceride) ratio for P. pelagicus early juveniles. The cholesterol experiment showed that out of 7 levels of dietary supplemental cholesterol (0, 2.5, 5.0, 7.5, 10, 12.5 and 15 g kg-1) tested, 10 g kg-1 consistently yield the best survival, development and growth with both the low and high end of cholesterol levels iv gave inferior development and growth results. In addition, the highest incidents of molt death syndrome (MDS), 20.7% were found for crabs fed the diet with the highest level of cholesterol supplementation at 15 g kg-1. PL is known to play an important role in lipid and carbohydrate metabolism as well as enhancing absorption of ingested fats, including triglycerides and cholesterol. For second experiment, six iso-lipidic diets with PL supplemented at 0, 30, 60, 90, 120 and 150 g kg-1 were tested and the results demonstrated that the diet without PL supplementation significantly impaired survival (with high incidents of post molt death), development and growth. PL supplemented at 120 and 150 g kg-1 yielded the best survival, development and growth that were often differed significantly to other treatments. Salinity stress test (salinity dropped abruptly form 30 to 7‰ for 7 h) using C4 crabs also showed that while the majority of crabs from PL 120 and 150 g kg-1 treatments survived to the end of stress test, all crabs from other treatments dead within 5 h. As interactive effects between cholesterol and PL have been demonstrated in other crustaceans, two experiments were designed to test this for P. pelagicus. In the first experiment, 10 iso-energetic diets were formulated, including 9 diets in a 3x3 factorial design with various combinations of 3 levels of PL (30, 60 and 90 g kg-1) and cholesterol (0, 5 and 10 g kg-1), respectively and a basal diet (deficient in both PL and cholesterol). It was demonstrated that the addition of PL at 90 g kg-1 could compensate lack of dietary cholesterol by substantially improved crab survival. As the first experiment showed that crabs fed the diet with the highest supplemented PL (90 g kg-1) and cholesterol (10 g kg-1) v yielded the best results, a second experiment was conducted to investigate if higher PL and cholesterol levels would lead to even better performance. Four diets with combinations of 2 cholesterol (10 and 15 g kg -1) and PL levels (90 and 120 g kg-1) were fed to C1 crabs and the results showed that the further increase in cholesterol and PL did not lead to significant improved survival, development and growth. With optimal cholesterol and PL level established, 3 experiments on neutral lipids requirements were executed. Firstly, effects of different levels of total neutral lipids (triglyceride) were assessed with 6 triglyceride levels at 0, 20, 40, 60, 80 and 100 g kg-1 (fish oil to corn oil ratio fixed at 2:1) being included in the diets. The results demonstrated that level of triglycerides did not significantly affect survival of the crabs (p>0.05), however, crabs fed the diet supplemented with 40 g kg-1 triglycerides achieved significant better growth than all other treatment except the diet supplemented with 20 g kg-1 triglycerides. The 40 g kg-1 triglycerides treatment also had the highest survival among all diet treatments. A subsequent experiment set triglyceride at the optimal level of 40 g kg-1 with varied fish oil (FO) to soybean oil (SO) ratio at 1:0, 3:1, 2:1, 1:1, 1:2, 1:3 and 0:1, respectively, to investigate their effects on performance of P.
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