Microalgae Schizochytrium sp . in Feed for Piau Leporinus friderici

1Laboratory of Aquaculture and Aquatic Ecology, Federal University of the Jequitinhonha and Mucuri Valleys, Highway MGT 367, km 583 Alto da Jacuba, no 5000, zip code 30100-000 Diamantina, MG, Brazil 2Laboratory of Nutrition of Aquatic Organisms, Federal University of Viçosa, Avenue Peter Henry Rolfs, w/n University Campus, Viçosa MG, 36570-900 Viçosa, MG, Brazil 3Laboratory of Animal Nutrition, Federal University of the Jequitinhonha and Mucuri Valleys, Highway MGT 367, km 583 Alto da Jacuba, no 5000, CEP 30100-000 Diamantina, MG, Brazil


Introduction
The species Leporinus friderici, Bloch 1794 have a great commercial value because of their wellappreciated meat (Nomura, 1984).Several types of research on incorporating essential nutrients in fish feeds are a new way to food enrichment for human consumption (Li et al., 2009;Qiao et al., 2014;Kousoulaki et al., 2015).
It is known that the fish body protein and lipid profile can be adjusted according to diet and inclusion of polyunsaturated acid (PUFA) source, such as Docosahexaenoic Acid (DHA), found in microalgae, being an alternative to changes organoleptic caractheristics and improve the growth (Lenihan-Geels et al., 2013;Martins et al., 2013).
A several species of microalgae, as Schizochytrium sp., are identified as rich in carbohydrates, proteins, lipids and nutritionally valuable components (Sarker et al., 2016;Sathasivam et al., 2017).A practical way of enriching diets for captive-bred fish is the inclusion of microalgae, which can modify the lipid and protein profile of the animal muscle composition (Richmond, 2004;Li et al., 2009;Qiao et al., 2014).Rsearches with freshwater fish shows increased lipids and protein profile after the inclusion of microalgae in feed, improving fish growth (Qiao et al., 2014;Sarker et al., 2016), been an alternative to increase productive yield.
Thus, the objective of this study was to evaluate the growth and the body chemical profile of L. friderici juveniles fed with diets supplemented with different levels of the microalgae Schizochytrium sp.

Material and Methods
The experiment was conducted indoors, in the city of Diamantina-MG, in Brazil (18°15' south latitude, 43°36' west longitude and 1.400 m above sea level), from September 12 to November 10 of 2015 (60 days).The research was carried out in accordance with the ethical standards and approved by the Ethics Committee on Animal Use (process n° 029/2016).
Prior to the experiment, fish were adapted to a trial for seven days, being fed a control artificial diet (without the inclusion of Schizochytrium sp.).Then, 140 juveniles were selected, weighed and measured (11.80±1.08 g and 9.68±0.31cm, respectively).These juveniles were stocked in 20 aquariums (35 L) at a density of 0.2 fish L −1 , or seven fish per tank.These aquariums were provided with aeration and controlled temperature.
An artificial feed was prepared and supplemented with 0, 10, 20, 30 and 40 g of Schizochytrium sp.kg −1 of feed (Table 1).All the diets were extruded (INBRAMAQ MX40) and beads were produced with a mean diameter of 2 mm.Each treatment had four replicates, in a completely randomized design.The juveniles of L. friderici were fed ad libitum three times a day (10 am, 1 pm and 4 pm).The composition of the microalgae meal is shown in Table 2.
The aquariums were cleaned three times a week (Monday, Wednesday and Friday) to remove waste.Weekly, water quality parameters were measured, such as temperature (°C), pH, dissolved oxygen (mg L −1 ) and conductivity (µs cm −1 ), using a multiparameter (U-50 Horiba).The concentrations of total ammonia, nitrite and nitrate (mg L −1 ) were measured according to (APHA, 2012) (method 4500).
Fish whole-body and artificial diet proximate compositions were determined using the standard methods of AOAC (2012) (methods: 990.03 protein, 2003.05lipids, 930.15 dry matter, 965.17 phosphorus 968.08 calcium) at the Laboratory of Animal Nutrition of the Department of Animal Science.In the whole-body analysis, dry matter, mineral matter, crude protein, lipids, calcium and phosphorus contents were determined.All the analyses were carried out in duplicates.
Means and standard deviations were calculated for water quality parameters and characterization of the culture environment.To evaluate the effects of the inclusion of Schizochytrium sp. in the fish diets, the growth parameters and body chemical composition data were analyzed by ANOVA and linear regression, using the SigmaStat 3.5 software (Systat Software Inc.).
No differences (p>0.05) were observed for dry matter, mineral matter and lipids of L. friderici juveniles (Table 5).Still, with the increase of Schizochytrium sp. in the diets, crude protein, calcium and phosphorus levels in juveniles had a linear decreasing effect (p<0.05).

Discussion
The water quality parameters were maintained constant throughout the experiment period, falling within the ranges recommended for tropical species (Boyd and Tucker, 2014) and for Leporinus species (Sipaúba Tavares and Magalhães-Santeiro, 2013), thus not compromising the development of L. friderici juveniles.
Weight gain, weight, biomass, feed intake, SGR and K increased as the amount of Schizochytrium sp.included in the diets of L. friderici juveniles was raised.Freshwater fishes inhabit environments that are poor in polyunsaturated fatty acids, mainly docosahexaenoic acid; therefore, through an evolutionary pressure, they are able to retain the content produced endogenously (Tocher, 2010), which explains the efficient assimilation of Schizochytrium sp. in this experiment.
Algae inclusion in feeds had no influence on juvenile total length and food conversion.Sarker et al. (2016) also observed a low feed conversion in tilapia fed the same algae species, but with no differences in total length.Likewise, river prawns are known to have a better feed conversion when supplemented with Schizochytrium sp., but again with no effect on total length (Kangpanich and Senanan, 2015).
The microalgae Schizochytrium sp. can enhance the efficiency of nutrient absorption by the gastrointestinal tract since its content of fatty acids improves digestion, which has contributed to L. friderici juvenile growth.Similarly, Sarker et al. (2016) noted that tilapia fed diets supplied with Schizochytrium sp. had an improvement in weight gain and weight.Likewise, Hoestenberghe et al. (2016) also observed a higher weight gain in jade perch juveniles (Scortum barcoo).According to Li et al. (2009), the addition of 1.0 g kg −1 of dried microalgae (Schizochytrium sp.) in the diet of channel catfish (Ictalurus punctatus) promoted weight gain when compared to control artificial diets (without the microalgae).Moreover, (Li et al., 2009;Santos et al., 2015) reported increases in weight gain and biomass when Schizochytrium sp. was added in the diets of Nile tilapia (Oreochromis niloticus) and catfish (I.punctatus), respectively.
The dietary inclusion of Schizochytrium sp.microalgae increased feed intake of L. friderici juveniles, as observed for Atlantic salmon juveniles (Salmo salar) by Kousoulaki et al. (2015).Conversely, tilapia juveniles had a decrease in feed intake with an increasing inclusion of the microalgae (Sarker et al., 2016).As in this study, channel catfish had a greater feed intake when Schizochytrium sp. was included in the feed (Li et al., 2009).
The SGR of L. friderici juveniles increased as the levels of microalgae was raised in the diets, as already observed for sea cucumbers and prawns fed diets supplied with Schizochytrium sp.(Kangpanich and Senanan, 2015;Md et al., 2017).Yet, for freshwater fishes, the addition of Schizochytrium sp. in the diet had no influence on SGR (Sarker et al., 2016;Qiao et al., 2014); yet the use of microalgae oil (Crypthecodinium cohnii and Schizochytrium sp.) in diets reduced SGR in gilthead sea bream (Sparus aurata) (Ganuza et al., 2008).Despite these reports, SGR studies in fish fed microalgae are still scarce.
Fulton's condition factor (K) is the ratio between body weight and length; it expresses the degree of well-being and feeding of fish in a previous season (N'da et al., 2016).This factor remained similar among the additional levels of Schizochytrium sp. in the diet, showing a linear effect, thus indicating that fish wellbeing was increased with the inclusion of this organism.
The values observed in this study were higher than those reported in the literature (Guidelli et al., 2011;Nascimento et al., 2012), suggesting that the culture conditions were adequate for L. friderici juveniles.According to Adite et al. (2017), relatively high condition factors, indicated by K factor, promoted a perfect establishment of Chrysichthys nigrodigitatus in the aquatic environment they were growing.
The L. friderici juveniles an improved dry matter and lipids levels in the whole-body when the supplementation of Schizochytrium sp. was increased and the level of protein was greater with the inclusion between 10 to 30 g of Schizochytrium sp.kg −1 in the feed.The increase of the corporal dry matter and protein, with the elevation of the levels of Schizochytrium sp. was directly related to L. friderici growth, with the increase in weight gain, biomass and SGR.
Sarker et al. (2016) also observed higher levels of body protein in juveniles of tilapia and consequently an increase in weight gain after feeding with Schizochytrium sp.The level of body protein also increased in juveniles of Paralichthys olivaceus fed with diets enriched with Schizochytrium sp. and Nannochloropsis sp.(Qiao et al., 2014).Juveniles of the channel catfish fed with diets enriched with Schizochytrium sp. had no significant difference in the contents of protein, lipids and moisture in fillet (Li et al., 2009), may be associated with the food habit of the species.Microalgae are a source of protein and lipids (Fleurence, 1999;Guccione et al., 2014) for fish species.
The increase of the body protein content in L. friderici, since proteins possess excellent amino acid scores and digestibility characteristics for humans, is a productive advantage and the enrichment of fish meat with lipids sources, as DHA, makes it a functional food for human health.
Contents of calcium in the body chemical profile of L. friderici decreased linearly with the inclusion of Schizochytrium sp. in the diet.Perhaps, the saponification reaction (Lehninger et al., 2008) between fatty acids in microalgae and this mineral in the gut of the juvenile impairing the digestion and metabolism of this mineral, hence causing the lower quantities found in the fish body composition.

Conclusion
Juveniles of Leporinus friderici fed with artificial diets supplemented with increasing levels of Schizochytrium sp. have better growth and changes in it the body chemical profile.

Table 1 :
Composition and analysis of experimental diets (natural matter) Treatments Schizochytrium sp.Alltech Inc. 2 Vitamin and commercial mineral supplement for fish; guarantee levels (per kg of product): vit.A,

Table 3 :
Water quality parameters during the experimental period, 56 days, of Leporinus.friderici juveniles fed with different levels of Schizochytrium sp.

Table 4 :
Performance of Leporinus friderici juveniles fed with diets supplemented with Schizochytrium sp., during 56 days -effects of linear and quadratic order concerning the inclusion of Schizochytrium sp. in the diet.

Table 5 :
Body chemical analysis of Leporinus friderici juveniles fed with diets supplemented with Schizochytrium sp., during 56 days Treatments