Effect of different rotifers enrichment formula feed on growth and survival of sea bream larvae ("Sparus aurata")

Isabel María Fernández Artiles Degree in Marine Science (ULPGC) [email protected] Academic year 2013/2014 Tutors: Carmen María Hernández-Cruz Lidia Robaina Robaina Final assignment for the Marine Science degree Effect of different rotifers enrichment formula feed on growth and survival of sea bream larvae (Sparus aurata) Isabel María Fernández Artiles 2 Index Acknowledgements 3 TABLE LIST 4 FIGURE LIST 5 ABBREVIATIONS 6 1. Introduction 7 1.1 Global situation of aquaculture 7 1.2 Larviculture 8 1.3 Objectives 11 2. Material and methods 12 3. Results 15 3.1 Growth 15 3.2 Survival rate 17 3.3 Biomass 17 3.4 Activity test 18 4. Discussion 19 5. Conclusions 22 6. References 23 Isabel María Fernández Artiles 3 Acknowledgments Desde esta página quisiera mostrar mi agradecimiento a todas aquellas personas que de una forma u otra tuvieron relación con la elaboración de este trabajo. En especial: A mi familia, sobretodo a mis abuelos, por su apoyo incondicional durante toda la carrera y la realización de este trabajo. A la Dra. Carmen María Hernández-Cruz, por su inestimable paciencia y por ayudarme a esforzarme cada día más. A Lidia Robaina por la ayuda brindada, a pesar de que estuviese ocupada, siempre estuvo ahí. A Ángelo Santana, por su ayuda en los momentos más difíciles de este trabajo. Sin él la estadística no sería lo mismo. A todos los técnicos del mesocosmos del Parque científico Tecnológico de Taliarte, por su aceptación como una más durante los casi tres meses que estuve allí. A Desi, por su inestimable ayuda y paciencia a la hora de llevar a cabo este experimento y por enseñarme a confiar más en mí misma y ser autosuficiente. A Mario, por su predisposición a ayudarme, siempre con una sonrisa. A mis amigos y compañeros de clase que han estado allí para darme ánimos en esta última etapa de la carrera, especialmente a Yvonne, Dani y Jesús. Isabel María Fernández Artiles 4 TABLE LIST Pages Table I. Larvae size, weight, biomass, survival rate and activity test along the experiment. 15 Isabel María Fernández Artiles 5 FIGURE LIST Pages Figure 1. Development of global aquatic production (aquaculture and fisheries) from 1951 to 2011 7 Figure 2. Average size of 11th DPH sea bream larvae fed with the different diets 16 Figure 3. Average size of 14th DPH sea bream larvae fed with the different diets 16 Figure 4. Average weight of 11th DPH sea bream larvae fed with the different diets 16 Figure 5. Survival rate (%) of sea bream larvae at the end of the experiment according to different diets. The diets with the same letters are not significantly different (p-value > 0.05) 17 Figure 6. Biomass (µg) of sea bream larvae at the end of the experiment according to different diets 18 Figure 7. Larval survival rate after activity test at the end of the experiment according to different diets 18 Isabel María Fernández Artiles 6 ABBREVIATIONS FAO APROMAR PUFAs EPA DHA UV GIA ANOVA DPH Food and Agriculture Organization of the United Nations Asociación Empresarial de Productores de Cultivos Marinos de España Polyunsaturated fatty acids Eicosapentanoic acid 20:5 (n-3) Docosahexanoic acid 22:6 (n-3) Ultraviolet Aquaculture Research Group Analysis of variance Days post hatch Isabel María Fernández Artiles 7 1. Introduction 1.1 Global situation of aquaculture Since the beginning of civilization, the extraction of marine resources through fisheries and aquaculture has been present. Some examples of this are found in Egypt or China where, since ancient times, fishes have been breeding naturally. Even now, you can find people taking care of fish in small pounds caused by the overflow of the Nile River in Egypt or in Chinese rice fields, where carps are also farmed with the harvest. (Rueda, 2011) Actually, the origin of the marine resources that we use is not only from fisheries, also aquaculture plays a crucial role. According to reports from the FAO in 2012, the increasing of fisheries has made that, about 30% of the fish stocks were found fully exploited. Also, the consumption per capita of seafood (excluding algae) has increased from 9.9 kg in 1960 to 19 kg in 2010. This has favored the boost of the aquaculture in the past three decades, having as a consequence an increase of the fish global consumption with 8.8% annual rate. This growth also is related with an increase of its global fish contribution from 9 - 49% in the last 30 years (APROMAR, 2013). Figure 1. Figure 1. Development of global aquatic production (aquaculture and fisheries) from 1951 to 2011. APROMAR 2013 Currently, considering only fish, rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar) and sea bream (Sparus aurata) are, in that order, the fish species widely cultured in Europe (APROMAR, 2013). Isabel María Fernández Artiles 8 1.2 Larviculture Sea bream (Sparus aurata) is one of the most produced fish in Spain (APROMAR 2013). It is mainly due for its ease of cultivation and also by its high growth rate (between 1.5 and 2 years to reach commercial size). However, and despite the high gamete production of this specie during the three months of breeding, just 90% of the eggs are fertilized and around 70% of these hatch successfully. Moreover, only among 20 - 35% of these larvae percentage survives (Ortega, 2009). Most of this high mortality rate occurs during transition among endogenous to exogenous feeding. According to different authors (Pascual & Yúfera, 1987; Rivera & Botero, 2009), the mortality occurrence is mainly due to larvae morphological limitation, involving mouth size, and also physiological restrictions as a result of an incomplete development of the digestive system. Through the years, a direct relationship between the type and quality of food provided and the extent of larval survival has been demonstrated (Lazo, 2000; Conceição et al., 2010; Hamre et al., 2013). The simplicity of digestive tract in hatched larvae, being little more than a straight tube without differentiation and with a very low enzyme activity (Govoni et al., 1986), makes elaborated diets inefficient at this stage of development (Lazo, 2000; Izquierdo et al., 2000; Conceição et al., 2010). Some authors such as Conceição et al. in 2010, not only point out to the digestive system of the larvae as the responsible of a poor diet. They also qualifies them as visual predators, being the movements of living prey the trigger of their feeding response. This predatory instinct in the stages of larval development supports zooplanckton living preys as the most appropriate diet for larval feeding. According to Cerecedo-Civera et al. in 2004, to ensure the supply of larvae by living prey; they must have certain requirements such as: having the right size to the larvae mouth, a slow movement, a high availability and also, they have to stimulate larvae hunting instinct. Besides, the preys must reach all minimum nutritional requirements and, therefore, with easily digestion for the larvae. Few species of zooplankton reach these requirements; among them we can find rotifers (Brachionus sp.), brine shrimp and copepods, being the first two species the most widely cultivated for the nutrition purpose (Lazo, 2000; Conceição et al., 2010). Isabel María Fernández Artiles 9 Some reasons make rotifers an excellent source of living prey for larvae during its first weeks of exogenous feeding: they have a small size, between 70 - 350 μm, a high growth rate, the capability to resist high population densities and an elevated tolerance range to growing conditions and management. The rotifers also have an elevated content of protein, between 29 - 63% of its dry weight (Jeeja et al., 2011). Nevertheless, only between 9 - 28% of its dry weight is lipid composition, which can vary according the diet supplied to the rotifers (Conceição et al., 2010). These lipids are polyunsaturated fatty acids (PUFAs), belonging to the group of phospholipids, which serve as the main energy source for marine fish larvae in their early stages (Rodriguez et al., 1997; Lazo, 2000; Bell et al., 2003; Hamre et al., 2013.). Among these fatty acids, eicosapentaenoic acid (EPA) 20:5 (n-3) and docosahexaenoic acid (DHA) 22:6 (n-3) plays a very important role. These PUFAs not only just provide energy to larvae; they are also essential for the cell membranes integrity and functions (Sargent et al., 1997; Rivera & Botero, 2009). Despite that, some studies show the limited capacity of fish larvae to convert EPA into DHA. Sargent et al. (1997) have demonstrated an important deficiency of the enzyme delta-5-desaturase, responsible for enlongation and desaturation of PUFAs. Besides, rotifers are usually fed with baker's yeast, which gives them a deficient composition of PUFAs that will be transmitted to the larvae by prey ingestion. So, to improve the uptake of these fatty acids, larvae are feed with PUFAs high enriched rotifers through microalgae or commercial products (Hamre et al., 2008; Conceição et al., 2010). This enrichment must have a 1:2 ratio of EPA:DHA according to Lazo (2000), who proved that, in excess, these PUFAs generate harmful effects on neuronal and optical system of the larvae. Along with a diet rich in PUFAs, an appropriate culture medium also promotes larval survival. The technique called "green water", which is based on the addition of microalgae directly to larval culture tanks, has achieved great results in terms of increased survival, growth and ingestion rate of larvae (Øie et al., 1997; Reitan et al., 1997). Rocha et al. (2008) also proved that the presence of microalgae in tanks increases the feeding response of larvae of sea bream significantly. Furthermore, the “green water” technique contribute to preserve the nutritional value of prey (Makridis & Isabel María Fernández Artiles 16 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Size (mm) DPH 11 Figure 2. Average size of 11th DPH sea bream larvae fed with the different diets. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Size (mm) DPH 14 Figure 3. Average size of 14th DPH sea bream larvae fed with the different diets. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Weight (μg) DPH 11 Figure 4. Average weight of 11th DPH sea bream larvae fed with the different diets. . Isabel María Fernández Artiles 17 3.2 Survival rate Regarding survival rates, the data obtained at the end of the experiment showed significant differences after perform statistical analysis (p-value = 0.013). These differences were found between diet 3 (Isochrysis sp.), which had the highest survival rate, and diets 1 (DHA Protein SELCO) and 4 (Dunaliella sp.), both with the lowest survival rate as is shown in figure 5. However, due to the abnormality of the diet 1 data, which should have been one of the best survival rates, it is thought that it could be a problem with the product. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Survival rate (%) Figure 5. Survival rate (%) of sea bream larvae at the end of the experiment according to different diets. The diets with the same letters are not significantly different (p-value > 0.05) 3.3 Biomass Once the experiment ended, all the tanks had a variable number of survival larvae for the count of the biomass and its subsequent statistical analysis. As we can see in the figure 6, its distribution is similar to the obtained for survival rate except that, in this case, the differences of larvae biomass found between diets were not significant. It was also shown that the highest biomass was found in the tanks where larvae were fed with diet 3 (167.57 mg) while the lowest were again in tanks whose larvae were fed with diets 1 (DHA Protein SELCO) and 4 (Dunaliella sp.), being the last one the diet with the lowest obtained biomass (Figure 6). a a b ab ab Isabel María Fernández Artiles 18 0 20 40 60 80 100 120 140 160 180 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Biomass (μg) Figure 6. Biomass (µg) of sea bream larvae at the end of the experiment according to different diets. 3.4 Activity test Observing the results of activity test (Figure 7), a similar distribution was found to those obtained for survival and biomass (Figures 5 and 6 respectively). It was moreover observed that larvae fed with diets 1 and 4 showed a decreasing survival rate after the applied stress, whereas larvae fed with diet 3 were the most are more resistant, with 86.67% of survival. 0 10 20 30 40 50 60 70 80 90 100 Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Survival rate (%) Figure 7. Larval survival rate after activity test at the end of the experiment according to different diets. Isabel María Fernández Artiles 19 4. Discussion Analyzing the results, we can’t see any concluding response between tested diets regarding the size of sea bream larvae at the samplings of the experiment. But with weight data obtained, we can see a slight trend toward higher weight in diet 5 (baker’s yeast). That trend might be related with low biomass levels found in the larvae tanks fed with that diet. This increase in larval growth associated with decreased biomass has also been described by Roo et al. in 2010. They tested larval growth at different densities, resulting in a higher growth rate at low densities (> 50 larvae L-1). So, they proved that high larval densities are associated with low rates of growth due to a decreased appetite, an increased aggressiveness of larvae and therefore an intense intraspecific competition for food resources. All this triggered a chronic stress situation in larvae, decreasing the biomass of the system and favoring the survival of larvae better adapted and more resistant (Hernandez-Cruz et al., 1999). However, the above described for biomass of diets 1 (DHA Protein SELCO) and 4 (Dunaliella sp.), where there are even less biomass in the tanks than in diet 5, does not correspond with the results obtained of average weight, which values should be higher. During this work, it has been exposed at many times the great importance of the intake of these fatty acids in the larvae. Many authors have found in them the response to an increase or decrease of growth and survival in these early stages of larval development (Salhi, 1997; Rodríguez et al., 1994; Salhi et al., 1994). The data obtained for diets 1 and 4, does not fit with the described by Hernández-Cruz et al. (1999) and Roo et al. (2010), showing then a poorly PUFAs enriched diet for larvae. Yeast supplied to rotifers in the diet 5, had around 1.3% of PUFAs [1] while the diet 4, made from the microalga Dunaliella sp., has only around 0.4 - 0.8% of these fatty acids (Nevejan et al., 2003). This agrees with the data observed for us. However, the data obtained for diet 1 do not correspond to expectations. A study by Silva in 1999 evaluated the content of PUFAs in rotifers enriched with DHA Protein SELCO, obtaining levels of EPA and DHA of 12.1 and 25.2 mg g-1 of dry weight of EPA and DHA respectively. Isabel María Fernández Artiles 20 These levels cannot be compared with those obtained for rotifers enriched with yeast which, according to Watanabe et al. in 1983, ranging from 1 to 2 mg g-1 of dry weight of EPA. This is why it’s thought that the anomalous data found in the diet 1 could be explained as a possible result of a fatty acid oxidation, perhaps due to a poor maintenance of the product. This would be clarified in part analyzing the biochemical composition of the diets, but that is not the objective of this experiment. On the other hand, we also have high levels of PUFAs in diet 3, made from Isochrysis sp., which do not show an increase in average larval growth compared with the other diets tested with lower content of these fatty acids. According Huerlimann et al. (2010), this microalgae has around 8.5 - 15.9% of dry weight of PUFAs. Silva in 1999 performed an experiment to analyze the level of PUFAs in Isochrysis galbana enriched rotifers, obtaining a composition between 15 - 20 mg g-1 of dry weight in EPA and DHA. These values are very similar to those obtained by this author in rotifers enriched with DHA Protein SELCO. However, for the tanks fed with diet 3, high biomass was observed, which were also reported by Hernandez-Cruz et al. (1999), and latter on by Roo et al. (2010). They stated that an increase biomass is associated with reduced growth of the larvae. However, neither the survival rate nor the larvae resistance were not reduced by this high biomass due to the high PUFAs levels in the diet. Similarly with the diet 3, the diet 2 (Tetraselmis sp.) exhibits low values of average weight and high in terms of biomass, survival and resistance. However, despite having relatively high levels of PUFAs (0.7 - 3.8% of dry weight of algae according Huerlimann et al. in 2010), this diet does not exceed the values achieved by diet 3 in any respect. Several authors argue that this may be due to the high requirements of DHA compared to EPA in larvae as a result of the high rate of growth and development of tissues and structures in the larval stage (Takeuchi et al., 1994; Rivera & Botero, 2009). DHA has been described as an important component of the tissue neural larvae and also improves vision and, consequently, better catch prey which leads to a better fed (Rodriguez et al., 1997; Furuita et al., 2000; Rivera & Botero, 2009). That is the reason why, despite that diet 3 has a lower content of EPA than diet 2, possessing a much higher value of the DHA improves larvae development. Isabel María Fernández Artiles 21 Finally, larvae fed with diets 2 and 3 showed a higher resistance to stress caused by the activity test, but larvae fed with diet 3 obtained the best results. An experiment conducted by Takeuchi et al. (1991) with larvae of Japanese sea bream (Pagrus major), showed that those fed larvae with a DHA rich diets performed better survival rate before the test activity than whose larvae which were fed with diets rich in EPA. Isabel María Fernández Artiles 22 5. Conclusions The growth of the larvae is highly dependent on the tank larvae density, and thus higher intensity of larvae competition during feeding. A reduction of number of larvae per tank appears a possible reduction to this problem. Among the tested diets, the use of microalgae Dunaliella sp. for rotifers enrichment does not provide acceptable results in larval growth and survival rate. By the other hand, the microalgae Tetraselmis sp., and despite its high levels of EPA, does not provide enough PUFAs for larvae mainly by its low DHA content. High DHA content diets, as that containing Isocrhysis sp. in present experiment, promote better survival and stress resistance of the larvae. Isabel María Fernández Artiles 23 6. References APROMAR (2013). La acuicultura en España 2013. APROMAR, ESACUA, Fundación OESA Bell, J. G., McEvoy, L. A., Estevez, A., Shields, R. J., & Sargent, J. R. (2003). Optimising lipid nutrition in first-feeding flatfish larvae. Aquaculture, 227(1), 211-220. Civera-Cerecedo, R., Alvarez-González, C. A., & Moyano-López, F. J. (2004). Nutrición y alimentación de larvas de peces marinos. Avances en nutrición acuícola VII. 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