Spirulina Productive performance and fatty acid profile of hungarian carp fingerlings fed with Spirulina enriched feed

The incentive for fish consumption by the population increased its captive breeding production and consequently, increased the demand for feeds. This study aimed at evaluating the use of Spirulina microalga as source of protein, in place of the Saccharomyces cerevisiae yeast, on the productive performance and fatty acid profile of Hungarian carp fingerlings (Cyprinos carpio). In order to carry out this experiment, a basal ration formulated with soybean meal and dehydrated yeast (Control), and other rations formulated with the Spirulina microalga substituting yeast at 25, 50, 75 and 100% levels. 280 Hungarian carp fingerlings with 50 days and average initial live-weight of 2.87 ± 0.02 g were used. The microalgae enriched rations increased linearly the weight gain and the specific growth rate of the fingerlings. The saturated Research, Society and Development, v. 9, n. 3, e116932301, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i3.2301 3 fatty acids represent more than 50% of the lipid content. In relation to the polyunsaturated fatty acids, an increasing rise was observed as the protein substitution of the ration by the microalgae increased.


Introduction
The world per capita consumption of fish increased from an average of 9.9 kg in 1960 to 17.0 kg in the 2000s to 18.9 kg in 2010. This increase has been as a result of the combination of population growth, income rise and urbanization associated to strong expansion of fish production and modern means of distribution (FAO, 2014).
Fish play an important role in global food security and in nutritional necessities of the people in developing and developed countries (FAO, 2014). Their nutritional benefits such as the high content of unsaturated fatty acids, as well as high biological proteins value, result in large participation of fish in the food market (Widjaja et al., 2009).
One of the most used protein sources in the preparation of rations for aquatic organisms is the soybean meal, however, this source presents antinutritional factors such as proteases inhibitors and lectins, and sulfur deficiency in the amino acids (Furuya et al., 2004). An Research, Society and Development, v. 9, n. 3, e116932301, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i3.2301 4 alternative ingredient in the substitution of soybean meal in fish feed is Saccharomyces cerevisiae yeast, which has a protein content ranging from 37 to 45% crude protein (Lazzari et al., 2007).
Another alternative for protein source substitution in feeds is the Spirulina microalgae which presents a high amount of protein and amino acid content, similar to those recommended by FAO (Food and Agricultural Organization). This microalga is considered as a source of carotenoids and essential fatty acids, with emphasis on gamma linolenic acid (Holman & Malau-Aduli, 2013). The biomass of the Spirulina microalgae has a GRAS (General Recognized as Safe) certificate, issued by the FDA (Food and Drug Administration) recognizing this microorganism safe for consumption, enabling it to be used in food, pharmaceutical and medical areas (Soccol et al., 2013;Mendonça et al., 2012). In addition, in the natural environment, microalgae serve as basis for feeding the fish larvae food chain as well as the zooplankton in the food chain and in aquaculture to feed fish, crustaceans and molluscs (Brown et al., 1997).
The fish as well as the rest of the animals are unable to endogenously produce -9, -6 e -3 fatty acids, therefore, they must be supplied by the feed. Although the influence of environmental parameters in determining lipid requirements for fish is important, feeding is the major factor that contributes to the fatty acid profile of these animals. Studies have shown that some farmed fish contain a greater amount of -3 fatty acids in their meat than in natural fish (Suárez-Mahecha et al., 2002).
This work had the objective of evaluating the utilization of Spirulina microalgae as protein source, in place of Saccharomyces cerevisiae, on the productive performance and the fatty acids profile of the carcasses of Hungarian carp fingerlings (Cyprinos carpio).

Elaboration of rations
The experimental diets were isoproteic, containing an average of 40% brutal protein.
Soybean meal associated to Saccharomyces cerevisiae or Spirulina sp. LEB-18 was used (Morais et al., 2008). The substitution of yeast by the microalgae took place at levels 0 (control), 25, 50, 75 and 100 % (Table 1). Research, Society and Development, v. 9, n. 3, e116932301, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i3.2301 5 To elaborate the formulations, the ingredients were first of all ground and sieved. The diets were mixed in a y-type mixer, pelleted and dried at 50 °C for 18 h in an air circulating dryer. After which, they were ground and sieved to obtain a diameter between 1.0 to 2.38 mm.

Biological tests
For biological tests, 280 Hungarian carp fingerlings (Cyprinos carpio) were used with 50 days and initial live weight of 2.87± 0.02 g, distributed in a completely distributed in a completely randomized design with 5 treatments and 4 replicates. The experiment lasted for 30 days and each experimental unit consisted of 20 aquariums with 50 L of water, each with an individual and constant aeration, containing 14 fingerlings.
Research, Society and Development, v. 9, n. 3, e116932301, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i3.2301 6 The animals were feed at a rate of 5% of the biomass, divided twice a day. The physical and chemical variables, being the temperature and dissolved oxygen (YFF-55 oximeter), pH (PHS-3B digital pH meter), total ammonia (Alfakit AT 10P photo colorimeter microprocessor) and alkalinity of water were evaluated volumetrically three times a week (ALPHA, 1998). It affected the daily cleaning of the bottom of the aquariums through syphoning and exchange of 30 % of water after the first feeding.

Fish body chemical composition analysis
At the end of the experimental period, 5% of the animals from each treatment were euthanized and frozen (-18 ºC). The analysis of the body chemical composition was performed with the whole fish, determining crude protein, lipids, ash and moisture (AOAC., 2012). The determination of the fatty acids profile was performed from the fish remains, using the method proposed by Bligh & Dyer (1959) for the extraction of total lipids. The methylation of fatty acids was performed by the method of Metcalfe et al. (1966). The determination of fatty acids was performed in a Varian -3400CX, a gas chromatograph equipped with a flame ionization detector and a fused silica column containing stationary phase of polyethylene glycol, 30 m long and 0.32 mm in diameter. The entrainment gas was nitrogen at 0.5 mL min -1 . The injector and detector temperatures were 250 and 280 °C, respectively. The initial column temperature was 100 °C followed by an increase of 8 °C min -1 to 230 °C, maintaining it constant for 20 minutes. Fatty acids were identified by comparing the retention times with standards and quantified by area normalization. The chemical composition and digestibility results of the rations were subjected to the variance analysis and tukey test, and for the data performance and fish body composition, ANOVA and regression analysis.
The ration with 100% protein substitution by the microalgae presented a low humidity (3.2%) in relation to the others. The lipid content was low in the control diet (3.2%) and the diets enriched with microalgae presented a growing increase in lipids content from 4.0 to 6.1%. The diets were isoprotein as expected, varying from 41.3 to 42.3% of protein, and the diets with low amount pf Spirulina sp. LEB-18 were more digestible since this microalga is easily digested because its cell wall is composed of mucopolysaccharides, simple sugars and proteins (Tomaselli, 1997). In relation to the body chemical composition, there was a reduction in the crude protein (p≤0,01) with an increase of the Spirulina flour level in the diet (Table 4). Similar results were Research, Society and Development, v. 9, n. 3, e116932301, 2020(CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i3.2301 8 observed by El-sayed (1994)
The polyunsaturated fatty acids varied from 4.3 to 38.5%, registering a growing increase of polyunsaturated fatty acids, with the increase of the feed protein substitution by microalgae.
According to the Department of Health of England, diets that present AGPI/AGS ratio above 0.45 (DHSS, 1984) and -6/-3 ratio below 4.0 (DH, 1994), are considered healthy under the human nutritional point of view. The fish subjected to treatment with 0, 25, 50, 57% yeast substitution with microalga presented an APGPI/AGS ratio below recommendation.
However, in ration to the -6 and -3, only the treatment with 75% yeast substitution by the microalga exceeded the recommended maximum.
In this work, only the fish subjected to 100% yeast substitution treatment with Spirulina microalgae presented values as recommended by the Department of Health of England, with AGPI/AGS of 0.66 and -6/-3 relation of 0.04, indicating that Spirulina sp. LEB-18 can constitute a nutritional source for the preparation of fish rations (Table 5).
The physical and chemical characteristics of water did not present significant variation between the treatments, having a mean temperature of 21 ± 1.2 ºC, dissolved oxygen 7.3 ± 1.2 mg/L, pH 7.1 ± 2, total ammonia 0.3 ± 0.1 mg/L and alkalinity 48 ± 5 mg of CaCO3/L, these being adequate conditions for the cultivation of common carp fingerlings according to Watanuki et al. (2006) and according to the recommended pisciculture limits (CONAMA, 2005). Makinouchi (1980) affirms that the best carp growth is within a temperature range of recommended by the author, it can be assumed that these temperatures did not influence negatively since behavioral observations, feed intake, growth and weight remained satisfactory throughout the experimental period.

Conclusions
The partial or total substitution of the yeast with Spirulina sp. LEB-18 microalga caused an increase in linear weight gain of the fingerlings, proving that the microalga concentrations in the rations used satisfy the necessities of the Hungarian fingerlings carp.
The saturated fatty acids represented more than 50% of the lipid content of the carcasses and for the polyunsaturated fatty acids, a growing increase was observed as the protein ration was substituted by microalga.
Considering that the lipid composition of fish reflects directly on the amount of their diet, the AGPI/AGS and -6/-3 ratios observed in the Hungarian carp fed with 100% Spirulina sp. LEB-18 ration indicate their nutritional value, making this microalga a protein source for the preparation of fish rations.