Effect of polyunsaturated fatty acids (PUFAs) on the proliferation and extracellular matrix mineralization and gene expression of gilthead seabream skeletal cell lines

The aquaculture industry aims at replacing significant amounts of marine fish oil by vegetable oils in fish diet. Dietary lipids have been shown to alter the fatty acid composition of bone compartments, which would impact the local production of factors controlling bone formation. Knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is however scarce in fish. Two in vitro bone-derived cell systems developed from seabream (an important species for aquaculture in the Mediterranean region) vertebra, capable of in vitro mineralization and exhibiting prechondrocyte (VSa13) and pre-osteoblast (VSa16) phenotype, were used to assess the effect of certain polyunsaturated fatty acids (PUFAs; arachidonic (AA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids) on cell proliferation, extracellular matrix (ECM) mineralization and gene expression. While all PUFAs promoted morphological changes in both cell lines, VSa16 cell proliferation appeared to be stimulated by PUFAs in a dose dependent manner until 100M, whereas proliferation of VSa13 cells was impaired at concentrations above 10M. AA, EPA and DHA inhibited VSa13 ECM mineralization, alone and in combination, while VSa16 ECM mineralization was only inhibited by AA and EPA. DHA had the opposite effect, increasing mineralization almost by 2 fold. When EFAs were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. Expression of marker genes for bone and lipid metabolisms has been investigated by qPCR and shown to be regulated in pre-osteoblasts exposed to individual PUFAs. Our results show that PUFAs are effectors of fish bone cell lines, altering cell morphology, proliferation and mineralization when added to culture medium. This work also demonstrates the suitability of our in vitro cell systems to get insights into mineralization-related effects of PUFAs in vivo and to evaluate the replacement of fish oils by vegetable oil sources in fish feeds.

Nutrition and farming practices as modulators of quality in gilthead seabream (Sparus aurata)

Gilthead seabream is the most important farmed species in the Mediterranean, and knowledge on how common farming practices impact its quality is limited. As such, this Thesis aimed to evaluate how gilthead seabream flesh quality is affected by some of these practices. In Chapter 2, the influence of nutritional factors was evaluated, specifically the high replacement of traditional marine-derived ingredients, both fishmeal and fish oil, with vegetable sources. We have seen that the vegetable-based diets tested did not greatly impact seabream flesh quality, although some alterations were seen in the fatty acid profile of the muscle. However, and despite having caused no alterations in flesh texture, vegetable ingredients reduced the amount of sulphated glycosaminoglycans in the extracellular matrix, affected muscle pH and reduced the activity of proteolytic enzymes. Throughout this Thesis, we measured for the first time the activity of proteolytic enzymes in seabream muscle, and cathepsin B was found to play a pivotal role in post-mortem muscle degradation. In Chapter 3, we evaluated the effect of harvesting and slaughter stress on seabream quality, and contrary to what is seen in most farmed species, our results show that gilthead seabream muscle structure is highly resistant to changes caused by stressful events. Nonetheless, considering that welfare is an increasingly important quality criterion, the use of a zero-withdrawal anaesthetic as a rested harvest technique or even slaughter method could prove valuable to the industry. In Chapter 4, we used maslinic acid as a dietary supplement, to modulate the muscle’s energetic status pre-mortem. As a finishing strategy, maslinic acid failed to increase levels of glycogen and ATP in the muscle. However, supplementation resulted in higher muscle fibre diameter and lower cathepsin B activity, and maslinic acid is likely to be useful to promote growth in this species. In general our Thesis has generated new knowledge to a major challenge facing the aquaculture industry, which is to find a compromise between the trends towards intensive rearing and consumer demand for healthy, high quality seafood being ethically acceptable and having a low impact on the environment.

Potential biomarkers on gametogenesis process and in sperm quality of european EEL (Anguilla anguilla)

Dissertação de mestrado, Aquacultura, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Development of Bioreactors for Nitrifying Water In Closed System Hatcheries Of Penaeid And Non-Penaeid Prawns

In the present study the development of bioreactors for nitrifying water in closed system hatcheries of penaeid and non-penaeid prawns. This work is an attempt in this direction to cater to the needs of aquaculture industry for treatment and remediation of ammonia and nitrate in penaeid and non-penaeid hatcheries, by developing nitrifying bacteria allochthonous to the particular environment under consideration, and immobilizing them on an appropriately designed support materials configured as reactors. Ammonia toxicity is the major limiting factors in penaeid and non-penaeid hatchery systems causing lethal and sublethal effects on larvae depending on the pH values. Pressing need of the aquaculture industry to have a user friendly and economically viable technology for the removal of ammonia, which can be easily integrated to the existing hatchery designs without any major changes or modifications. Only option available now is to have biological filters through which water can be circulated for the oxidation of ammonia to nitrate through nitrite by a group of chemolithotrophs known as nitrifying bacteria. Two types of bioreactors have been designed and developed. The first category named as in situ stringed bed suspended bioreactor(SBSBR) was designed for use in the larval rearing tanks to remove ammonia and nitrite during larval rearing on a continuous basis, and the other to be used for nitrifying freshly collected seawater and spent water named as ex situ packed bed bioreactior(PBBR). On employing the two reactors together , both penaeid and non-penaeid larval rearing systems can be made a closed recirculating system at least for a season. A survey of literature revealed that the in situ stringed bed suspended reactor developed here is unique in its design, fabrication and mode of application.

“Development of lymphoid cell culture system from Penaeus monodon and molecular approaches for its transformation

Unveiling the molecular and regulatory mechanisms that prevent in vitro transformation in shrimp remains elusive in the development of continuous cell lines, with an arduous history of over 25 years (Jayesh et al., 2012). Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. In addition, the regulatory mechanisms that prevent in vitro transformation and carcinoma in shrimps might provide new leads for the development of anti-ageing and anti-cancer interventions in human (Vogt, 2011) and in higher vertebrates. This highlights the importance of developing shrimp cell lines, to bring out effective prophylactics against shrimp viruses and for understanding the mechanism that induce cancer and ageing in human.. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. With this backdrop, the research described in this thesis attempted to develop molecular tools for induced in vitro transformation in lymphoid cells from Penaeus monodon and for the development of continuous cell lines using conventional and novel technologies to address the problems at cellular and molecular level.

Activated packed bed bioreactor for rapid nitrification in brackish water hatchery systems

A packed bed bioreactor (PBBR) was developed for rapid establishment of nitrification in brackish water hatchery systems in the tropics. The reactors were activated by immobilizing ammonia-oxidizing (AMONPCU- 1) and nitrite-oxidizing (NIONPCU-1) bacterial consortia on polystyrene and low-density polyethylene beads, respectively. Fluorescence in situ hybridization demonstrated the presence of autotrophic nitrifiers belong to Nitrosococcus mobilis, lineage of b ammonia oxidizers and nitrite oxidizer Nitrobacter sp. in the consortia. The activated reactors upon integration to the hatchery system resulted in significant ammonia removal (P\0.01) culminating to its undetectable levels. Consequently, a significantly higher percent survival of larvae was observed in the larval production systems. With spent water the reactors could establish nitrification with high percentage removal of ammonia (78%), nitrite (79%) and BOD (56%) within 7 days of initiation of the process. PBBR is configured in such a way to minimize the energy requirements for continuous operation by limiting the energy inputs to a single stage pumping of water and aeration to the aeration cells. The PBBR shall enable hatchery systems to operate under closed recirculating mode and pave the way for better water management in the aquaculture industry.

Establishment of Shrimp Cell Lines: Perception and Orientation

Development of continuous shrimp cell lines for effective investigation on shrimp viruses remains elusive with an arduous history of over 25 years. Despite presenting challenges to researchers in developing a cell line, the billion dollar aquaculture industry is under viral threat. Advances in molecular biology and various gene transfer technologies for immortalization of cells have resulted in the development of hundreds of cell lines from insects and mammals, but yet not a single cell line has been developed from shrimp and other marine invertebrates. Though improved growth and longevity of shrimp cells in vitro could be achieved by using modified growth media this did not make any leap to spontaneous transformation; probably due to the fact that shrimp cells inhibited neoplastic transformations. Oncogenic induction and immortalization are considered as the possible ways, and an exclusive medium for shrimp cell culture and an appropriate mode of transformation are crucial. In this review status of shrimp cell line development and its future orientation are discussed

Growth and nutrient utilization of Murray cod Maccullochella peelii peelii (Mitchell) fingerlings fed diets with varying levels of soybean meal and blood meal

The Australian native freshwater fish Murray cod, Maccullochella peelii pellii (Mitchell), currently supports a fledgling inland aquaculture industry, which is thought to have considerable growth potential. The aim of this study was to evaluate the suitability of two alternate protein sources [blood meal (BM) and defatted soybean meal (SBM)] as substitutes for fish meal at various levels of inclusion in diets for juvenile Murray cod. The growth performance of juvenile Murray cod in response to nine isonitrogenous and isocalorific diets (50% protein, 14% lipid, 20.2 kJ g⁻¹) consisting of a control diet in which protein was supplied from fish meal, and test diets in which the fish meal protein was substituted at levels of 8%, 16%, 24%, and 32% with BM or SBM was evaluated from a 70-day growth experiment. The per cent apparent dry matter (% ADC_dm) and percentage protein digestibility (% ADC_p) of the test diets were also determined using Cr₂O₃ as a marker. Survival in all the SBM dietary treatments was high but that of fish on the BM dietary treatments was significantly (P < 0.05) lower than in all the other dietary treatments. Specific growth rate (% day−1) of Murray cod fed SBM incorporated diets ranged from 1.63 ± 0.06 to 1.78 ± 0.10 and even at the highest level tested (32% of the dietary protein from SBM) was not significantly different (P > 0.05) from the fish fed the control diet (1.65 ± 0.09). Feed conversion ratios of the SBM dietary treatments ranged from 1.36 ± 0.08 to 1.45 ± 0.07. The protein efficiency ratios and protein conversion efficiencies of Murray cod in the soybean meal treatments were also good and for a majority of the SBM diets were better than those for the control diet. Per cent ADC_dm and ADC_p of the SBM diets tested ranged from 70.6 ± 1.46 to 72.3 ± 1.81% and 88.6 ± 0.57 to 90.3 ± 0.17%, respectively, and was not significantly different (P > 0.05) from the control diet (% ADC_dm 74.3 ± 1.63; % ADC_p 91.3 ± 0.55). The reasons for significantly poor survival and growth of Murray cod reared on BM incorporated diets, and relatively poor digestibility of these diets are discussed. The study shows that for Murray cod diets in which fish meal protein is substituted up to 32% performance or carcass composition is not compromised.

Allozyme diversity in Australian rainbow trout, Oncorhynchus mykiss

Rainbow trout, Oncorhynchus mykiss (Walbaum), were first introduced into Australia over 100 years ago, and forms the basis of important recreational inland fisheries and an aquaculture industry in south-eastern Australia. This paper investigates the genetic variation within and between samples of Australian rainbow trout using allozyme electrophoresis. The levels of genetic diversity within Australia do not show marked differences from those observed in hatchery and wild populations from throughout North America, New Zealand and South Africa, but there is evidence for the loss of some rare alleles during translocation from California to Australia via New Zealand. No appreciable difference in genetic diversity was apparent between hatchery and self-sustaining wild populations of rainbow trout from mainland Australia. However, significant differences in allelic frequencies were observed, with consistent genetic differences between Victorian and New South Wales samples most likely reflecting state-based hatchery and stocking policies.

Effects of dietary oil source on growth and fillet fatty Acid composition of Murray cod, Maccullochella peelii peelii

The Murray cod, an Australian native freshwater fish, supports a relatively small but increasing aquaculture industry in Australia. Presently, there are no dedicated commercial diets available for Murray cod; instead, nutritionally sub-standard feeds formulated for other species are commonly used. The aim of the present investigation was to assess the suitability of two plant based lipid sources, canola oil (CO) and linseed oil (LO), as alternatives to fish oil for juvenile Murray cod. Five iso-nitrogenous, iso-calorific, iso-lipidic semi-purified experimental diets were formulated with 17% lipid originating from 100% cod liver oil (FO), 100% canola oil, 100% linseed oil and 1 : 1 blends of canola and cod liver oil (CFO) and 1 : 1 blends of linseed and cod liver oil (LFO). Each of the diets was fed to apparent satiation twice daily to triplicate groups of 50 Murray cod with initial mean weights of 6.45 ± 1.59 g for 84 days at 22 °C. Final mean weight, specific growth rate and daily feed consumption were significantly higher for the FO and LFO treatments compared to the LO treatment. Feed conversion and protein efficiency ratios were not significantly different amongst treatments. Experimental diets containing vegetable oil and vegetable oil blend(s) had significantly higher concentrations of n-6 fatty acids, predominantly in the form of linoleic acid (LA), while n-3 fatty acids were present in significantly higher concentrations in LO and LFO treatments. The fatty acid composition of Murray cod fillet was reflective of the dietary lipid source. Fillet of fish fed the FO was highest in EPA (20:5n-3), ArA (20:4n-6) and DHA (22:6n-3). Fish fed the CO diet had high concentrations of oleic acid (OlA) (192.2 ± 10.5 mg g lipid− 1), 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). The present study suggests that fish oil 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.

Effect of crude oil extracts from trout offal as a replacement for fish oil in the diets of the Australian native fish Murray cod Maccullochella peelii peelii

The efficacy of trout oil (TO), extracted from trout offal from the aquaculture industry, was evaluated in juvenile Murray cod Maccullochella peelii peelii (25.4-0.81 g) diets in an experiment conducted over 60 days at 23.7-0.8 °C. Five isonitrogenous (48% protein), isolipidic (16%) and isoenergetic (21.8 kJ gm1) diets, in which the fish oil fraction was replaced in increments of 25% (0-100%), were used. The best growth and feed efficiency was observed in fish fed diets containing 50-75% TO. The relationship of specific growth rate (SGR), food conversion ratio (FCR) and protein efficiency ratio (PER) to the amount of TO in the diets was described in each case by second-order polynomial equations (P<0.05), which were: SGR=-0.44TO2+0.52TO+1.23 (r2=0.90, P<0.05); FCR=0.53TO2-0.64TO+1.21 (r2=0.95, P<0.05); and PER=-0.73TO2+0.90TO+1.54 (r2=0.90, P<0.05). Significant differences in carcass and muscle proximate compositions were noted among the different dietary treatments. Less lipid was found in muscle than in carcass. The fatty acids found in highest amounts in Murray cod, irrespective of the dietary treatment, were palmitic acid (16:0), oleic acid (18:1n-9), linoleic acid (18:2n-6) and eicosapentaenoic acid (20:5n-3). The fatty acid composition of the muscle reflected that of the diets. Both the n-6 fatty acid content and the n-3 to n-6 ratio were significantly (P<0.05) related to growth parameters, the relationships being as follows. Percentage of n-6 in diet (X) to SGR and FCR: SGR=-0.12X2+3.96X-32.51 (r2=0.96) and FCR=0.13X2-4.47X+39.39 (r2=0.98); and n-3:n-6 ratio (Z) to SGR, FCR, PER: SGR=-2.02Z2+5.01Z-1.74 (r2=0.88), FCR=2.31Z2-5.70Z+4.54 (r2=0.93) and PER=-3.12Z2-7.56Z+2.80 (r2=0.88) respectively. It is evident from this study that TO could be used effectively in Murray cod diets, and that an n-3:n-6 ratio of 1.2 results in the best growth performance in Murray cod.

Fish oil replacement in finfish nutrition

Unsustainable fishing practices have placed a heavy emphasis on aquaculture to meet the global shortfalls in the supply of fish and seafood, which are commonly accepted as the primary source of health-promoting essential omega-3 (n-3 highly unsaturated fatty acids). However, dietary fish oil is required for the production of omega-3-rich farmed fish and this commodity, in a vicious circle, is at present derived solely from wild fisheries. Decreasing global availability coupled with the highly variable price of this resource has forced the aquaculture industry to investigate the possibilities of alternative dietary lipid sources. This review attempts to compile all principal information available regarding the effects of fish oil replacement for the diets of farmed finfish, analysing the findings using a comparative approach among different cultured fish species. The review initially focuses on the present situation with regard to the production, availability and main nutritional characteristics of fish oil and the principal alternative lipid sources (such as vegetable oils and animal fats). Following this, the effects of fish oil replacement in finfish nutrition on feed quality, fish performance, feed efficiency, fish lipid metabolism, final eating quality and related economic aspects are presented and discussed.

Biology and management of Murray cod reproduction within controlled environments

The emerging Murray cod aquaculture industry is currently restricted by a lack of consistent and year-round production of fingerlings. Biological information and reproductive technologies developed during this research have facilitated the development of indoor, environmentally-controlled breeding practices for this species, thereby enabling Murray cod juveniles to be produced on-demand.

Leptin and lipid metabolism in fish

The aim of this chapter is to highlight potential links between leptin and the lipid metabolism in fish, within the context of current research interests of the fish nutrition amd aquaculture sector. In fact, it is envisaged that the whole aquacuture and fish biology sector will significantly benefit from an increased knowledge of such a powerful hormone, and it is believed this will contribute in transforming current aquaculture industry into an environmental and economical means of producing nourishing fish and seafood for the growing global population. The context is framed by a brief introduction on the current challenges in aquaculture and fish nutrition, and susequently the chapter focuses on the potential roles of leptin on voluntary food intake and lipid digestion and absorption in fish. Following this, the possible roles of leptin on lipid deposition and utilisation and the fatty acid metabolism in fish are presented and discussed. Eventually the possible interrelationships between dietary lipids and leptin are analysed underlining how knowledge gained from human nutrition could be extremely useful to tentatively address current constraints and obstacles found in the aquaculture nutrition sector. The present analysis suggests that we are likely only able to see the tip of the iceberg, and great and significant breakthrough discoveries will be likely by fathoming such fascinating area of research.

Δ-6 desaturase substrate competition : dietary linoleic acid (18∶2n-6) has only trivial effects on α-linolenic acid (18∶3n-3) bioconversion in the teleost rainbow trout

It is generally accepted that, in vertebrates, omega-3 (n-3) and omega-6 (n-6) poly-unsaturated fatty acids (PUFA) compete for ?-6 desaturase enzyme in order to be bioconverted into long-chain PUFA (LC-PUFA). However, recent studies into teleost fatty acid metabolism suggest that these metabolic processes may not conform entirely to what has been previously observed in mammals and other animal models. Recent work on rainbow trout has led us to question specifically if linoleic acid (LA, 18:2n-6) and ?-linolenic acid (ALA, 18:3n-3) (?-6 desaturase substrates) are in direct competition for access to ?-6 desaturase. Two experimental diets were formulated with fixed levels of ALA, while LA levels were varied (high and low) to examine if increased availability of LA would result in decreased bioconversion of ALA to its LC-PUFA products through substrate competition. No significant difference in ALA metabolism towards n-3 LC-PUFA was exhibited between diets while significant differences were observed in LA metabolism towards n-6 LC-PUFA. These results are evidence for minor if any competition between substrates for ?-6 desaturase, suggesting that, paradoxically, the activity of ?-6 desaturase on n-3 and n-6 substrates is independent. These results call for a paradigm shift in the way we approach teleost fatty acid metabolism. The findings are also important with regard to diet formulation in the aquaculture industry as they indicate that there should be no concern for possible substrate competition between 18:3n-3 and 18:2n-6, when aiming at increased n-3 LC-PUFA bioconversion in vivo.