Effect of spirulina ( Arthrospira platensis ) supplementation on tilapia ( Oreochromis niloticus ) growth and stress responsiveness under hypoxia

Several recent studies have shown that Arthrospira sp. supplementation of feeds has a beneficial effect on fish health and growth, but less is known about its possible effects on stress responsiveness. The present study was designed to evaluate using Arthrospira platensis as a feed supplement for Oreochromis niloticus fry, reared in recirculating aquaculture systems. Two isocaloric and isonitrogenous fishmeal-based diets were prepared with 0% and 1% A. platensis and fed to fry, approximately 10 mg live weight at the beginning of the experimental period (n=16 tanks, 8 tanks per treatment), at a feeding rate of 6% live weight, four meals a day for 50 d. The weight gain per tank, specific growth rate and feed conversion ratio were similar among treatments but A. platensis supplementation significantly increased survival ( p <0.05). Stress responsiveness was measured in all fish from 12 tanks using a non-invasive two-choice test. All fish from one home tank (n=6 tanks per treatment), were placed into a shaded tank where oxygen levels were slowly reduced. A doorway was then opened to a second illuminated tank with normal oxygen levels and the number of fish that left the home tank were counted. The fish fed A. platensis stayed significantly ( p =0.001) longer in the home tank (30.20 min ± 13.22) than controls (17.35 min ± 8.32), suggesting a lower stress responsiveness and a higher tolerance to hypoxia. used: CTR (control fish); FCR SPR ( Arthrospira platensis supplemented fish).


Introduction
Aquaculture provides more than half of the world's fish (FAO, 2016) but better feeding strategies are needed in order to keep it sustainable.One option is to use natural feed additives instead of artificial antibiotics, which cannot be used as growth promoters in the European Union (EC, 2003) since they promote microbial resistance.Arthrospira platensis is a natural product that can improve the quality of fish production by providing a rich source of vitamin B 12 and β-carotene (20 times more than carrots), essential amino acids, fatty acids (ω3 and ω6), minerals (James et al., 2006), protein (approx.65% dry weight; Phang et al., 2000), as well as anti-oxidant properties.For all of these reasons, it is commonly used as a natural supplement in animal feeds, including for fish (McCarty, 2007).
Less is known about how A. platensis can improve the condition of weaker fish or their response to stress.
Several studies have considered the effects of A. platensis supplementation (less than 10% of diet) in fish.Red swordtail (Xiphophorus helleri) given 8% A. platensis increased feed consumption, body weight, length, gonad weight and number of offspring (James et al., 2006).In three-spot gourami (Trichopodus trichopterus), Khanzadeh et al. (2016) found that 5% A. platensis (replacing fishmeal) improved feed intake, feed conversion ratio (FCR) and the gonadosomatic index.Rainbow trout (Oncorhynchus mykiss) fed with 10 % A. platensis (a natural pigment source) 2 deposit more carotenoids in muscle tissue (Teimouri et al., 2013).Adding A. platensis to feed for guppies (Poecilia reticulata) improved their tolerance to a toxin (methyl red), manifested by a noticeable reduction in the cytotoxic effects on red blood cells (Sharma et al., 2005).In Oreochromis niloticus, adding 0.5, 1 or 2% A. platensis to feed improves its immune system (Abdel-Tawwab & Ahmad, 2009), has antioxidant effects and promotes growth (Takeuchi et al., 2002), but less is known about its possible effect on behaviour under stressful situations.
Regarding possible behavioural effects, A. platensis contains many different nutrients, including tryptophan and B6, which decrease psychological distress in humans (Shor-Posner et al., 1994).Fatty acids ω3, also present in A. platensis, can inhibit adrenal activation elicited by a mental stress in dogs (Delarue et al., 2003).Thus, supplementing O. niloticus diet with A. platensis could change their stress responsiveness.In their natural habitat, O. niloticus normally consumes algae (including cyanobacteria) and some invertebrates (Ibrahem et al., 2013).
In the past decade, there have been many studies on fish welfare (Huntingford et al., 2006), and several methods have been proposed to measure stress, including non-invasive behavioural methods to measure stress coping styles using a two-choice test (Laursen et al., 2011).Barreto & Volpato (2011) found that O. niloticus can be sorted into two opposing coping styles under stress, namely proactive (dominant) or reactive (passive).It is assumed that the development of a coping style depends on the genetic makeup of the fish and its previous experience (Sørensen et al., 2013), but less is known about possible effects of nutrition.In this study we set out to verify whether a 1% A. platensis supplementation affected growth and survival as well as the behavioural response to stress using the two-choice test.

Algae culture
We used the A. platensis strain PCC 9108 obtained from the Culture Collection at the Spanish National Research Council (CSIC).A. platensis was grown in plastic bioreactors (120 × 12 cm) in a greenhouse at the Universidad Politécnica de Madrid (40.446353N, -3.738341 E), from July to August using the natural photoperiod.The cultivation media was composed of four solutions (Table 1) modified from Zarrouk (1966).A. platensis was harvested when the culture reached approx. 1 g/L of dry weight.The harvest was performed from 9:30 to 10:30 am when the protein content was higher (Jourdan, 1999).Then the cells were dried on a horizontal sheet at 50ºC for approx.4-6 h and kept in an opaque container at 4°C to prevent oxidation before inclusion in the fish feed.

Fish and feeds
O. niloticus fry originally purchased from Valenciana de Acuicultura (Puçol, Valencia), were housed in 16 green fiberglass tanks (120 L, 0.46 m high, and 0.64 m in diameter).Each tank was connected to a filter (EHEIM Classic; MOD.2217, 6 L of capacity, 20 W and water flow 1000 L/h) and aerated with an air pump common to all tanks.This set up allowed us to maintain independent water conditions in each tank so as not to mix sediments and bacteria among treatments.The filters were prepared by inoculating nitrifying bacteria Solutions A and B were added to the macroelement solution at the rate of 1 mL/L, and the trace solution at 100 mL/L.
(EHEIM water care), using the supplier indications.
We added 5 ppm of ammonia daily for 3 weeks, testing the evolution of nitrogen compounds, until microbial activity was satisfactory.The distribution of treatments among the tanks was random.
O. niloticus fry were introduced in the tanks randomly and given a two-week acclimatization period.After that, all the fish from each tank were weighed in bulk and their length measured individually using a ruler (day 0).The initial average weight per tank was a 2.64 ± 0.45 mg (mean ± SD; n= 25) and initial fish total length was 1.17 ± 0.10 cm, with no significant differences among treatments.Fish were counted and weighed in bulk at 30 d, and counted, weighed and measured individually at 50 d.To quantify mortality during the fry phase, tanks were checked every day and dead fish were counted and removed.Mortalities were not concentrated on any particular day nor in any particular tank.
To make the experimental diet, commercial feed (Skretting T3) was crushed and sifted to a crumb size of 0.5-1 mm, mixed with 1% of A. platensis by weight and passed through the same sieve.The control diet was made in the same way but with no added algae.To analyze the proximal composition of the feed, five samples were taken per treatment and the percentage of dry matter (DM) was obtained by oven drying at 105ºC, to constant weight, and the ash content by incineration at 550ºC.Protein content was measured by Kjeldahl analysis and the ether extract by hydrolysis.The fiber content was obtained by the Van Soest et al.´s (1991) method and finally the energy was calculated using a bomb calorimeter.Both feeds were isocaloric and isoproteic and were kept at 4ºC until used (see Table 2).Fish were fed at 6% live weight using an auto-feeder (EHEIM 3581) that provided four meals a day (at 7:00, 12:00, 17:00 and 22:00 h), beginning the trial on 04 Aug until 24 Sept 2015, for a total of 50 days (7 weeks).The filters were turned off for 20 min after each meal to avoid feed loss.

Two-choice test
At the end of the trial (50 d), we performed a twochoice test based on Laursen et al. (2011), built using two circular tanks that were identical to the growout tanks (120 L) and attached to one another via a closable gate, 16 cm in diameter (Fig. 1).Tank A was tightly covered with black plastic (shaded) and Tank B was illuminated (natural light).Twelve two choice tests were carried out, using six tanks per treatment, discarding the tanks with less than 20 remaining fry (for A. platensis fry, where all tanks had over 20 fish, the six tanks were chosen randomly).For each test, all the fish in a tank were placed in Tank A at the

Discussion
The main goal of fish nutrition is to provide a balanced mixture of ingredients to support vital functions at an acceptable cost.Following this line of thought, at the moment it would not be cost-effective to use A. platensis (≈40 €/kg) as a protein substitute, but it may be useful as a nutritional supplement since it improves feed efficiency, carcass quality, and physiological response to stress in several species of fish (Takechi et al., 2002;Abdel-Tawwab & Ahmad, 2009;Velasquez et al., 2016).
In the current study, adding 1% A. platensis to feed did not affect production indices (FCR, growth) compared to controls.This contradicts previous studies such as James et al. (2006), using Xiphophorus helleri (8% supplementation) and Abdel-Tawwab & Ahmad (2009) (0.5% to 1% supplementation) who observed improved growth.However, Takeuchi et al. (2002) found that the specific growth rate and feed efficiency of Arthrospira sp.fed Orechromis sp. were lower than that of controls.Our same time and left to acclimate for 30 min.Then the door to the second tank (Tank B) was opened, and Tank A was bubbled with nitrogen gas to decrease oxygen levels (at a rate of 1 ppm per 10 min).Tank B was oxygenated to maintain normal oxygen levels (approx.7 ppm) and a counter-current water flow was set up in each tank to avoid water flowing between tanks.Oxygen levels were monitored in both tanks every 5 min.Each test was recorded with a video camera to avoid the effects of any human disturbance.We noted the times when any fish left Tank A. The test ended when the first five fish had left Tank A or after one hour had passed.Then we measured the length of the fish in Tank B.

Statistical analysis
All statistical tests were carried out using R.commander (R Core Team, 2013).According to a Shapiro/Wilk test, the data on growth and survival were normally distributed and were analyzed using a one-way ANOVA.The data from the two-choice tests did not follow a normal distribution and were analyzed using a Kruskal-Wallis test.The level of significance was p<0.05 in both cases.

Fish growth and survival
There were no significant growth differences among treatments after 30 or 50 d.Total body length was not significantly different among treatments (cm; mean ± SD; 4.13 ± 1.09 control fish, CTR, 4.22 ± 0.97 A. platensis supplemented fish, SPR), nor the coefficient of variation in total length (mean ± SD; 0.26 ± 0.08 CTR, 0.28 ± 0.08 SPR).However, there were significant differences regarding mortality (p>0.05), which was lower in fry that received 1% of A. platensis (Table 3).

Two-choice test
The coping styles of the tilapia were significantly different between treatments (p>0.05).More SPR fry adopted a proactive coping style, taking a significantly longer time to leave Tank A, despite decreasing oxygen levels (Fig. 2).The CTR fish tended to leave Tank A in groups of two or more fish, while SPR fry tended to leave one by one (p>0.05;see Table 4).Also, focusing on fish size, it appeared that fry that left Tank A were smaller, less than 3 cm long in both treatments, but the difference was not significant (p=0.148)Values followed by different superscript letters in the same row are significantly different (p<0.05).
findings of a 9% higher survival rate are in accordance with other studies that report 6% higher survival at 1% Arthrospira sp.supplementation after a bacterial challenge (Ibrahem et al., 2013).However, Abdel-Tawwab & Ahmad (2009), and Ungsethaphand et al. (2010) did not observe differences in survival among fish fed with Arthrospira sp.compared to controls.Our study may be different, however, due to the size of the fish.When the trial began, tilapias were in the fry stage (around 2-3 cm body length), when they begin to change from being more omnivorous to carnivorous, at about 6-7 cm.After that they change back again to more phytoplanktivorous filter feeding (Ibrahem et al., 2013).The change to carnivorous feeding could increase hierarchical stress and cannibalism, reducing the survival of weaker fry (Berrios & Snow, 1983).In this scenario, the high vitamin content of A. platensis, among other possibilities, could have played a role, helping to promote growth and decrease mortality.

Two-choice test
Regarding the two-choice test, our results suggest that more of the fry consuming A. platensis supplemented feed adopted a proactive coping style, since more of them remained in a stressful environment despite the possibility of escape.As suggested by Barreto & Volpato (2011) the fish that remain in the "stressful" tank have a lower reaction to stress and can be classified as proactive.Although several recent studies have assessed coping styles in fish (e.g., Ferrari et al., 2016), few have considered the effects of nutritional supplementation.Some authors have shown that A. platensis affects plasma cortisol and glucose levels.In rainbow trout (Oncorhynchus mykiss) cortisol and glucose significantly decreased with increasing levels of inclusion of A. platensis at 0 to 10% (Yeganeh et al., 2015).In great sturgeon (Huso huso), Adel et al. (2016) found that blood glucose increased at a higher inclusion of A. platensis (10%).Furthermore, according to Delarue et al. (2003), adding ω3 fatty acids to fish diets can inhibit adrenal activation elicited by psychological stress.Arthrospira sp. has a high level of unsaturated long chain fatty acids such as ω3 and ω6, which may have provided a similar effect in O. niloticus.In this sense, Anzola (2013) found a decrease in psychological stress with higher tryptophan intake in dogs, and Shor-Posner et al. (1994) found the same effect in humans with tryptophan and vitamin B6 supplementation.A. platensis contains tryptophan (1.5% DM) and vitamin B6 (0.8% DM).Taking into account that the tryptophan requirement of O. niloticus is 1% of the diet, with the inclusion of 1% A. platensis, it was increased to 1.15%.The vitamin B 6 requirements in juvenile Oreochromis sp. are 15 to 16 ppm of the diet (Shiau & Hsieh, 1997), but in the current study that was increased to 80 ppm (500% more than estimated control levels) by adding 1% of A. platensis.
In fish, body length is a strong predictor of individual positions in a hierarchy (Ward et al., 2006).Large fish are presumably more proactive, dominating other individuals, taking prime feeding sites and aggressively excluding smaller subordinate competitors (Webster & Hixon, 2000).In our study, although it appeared that smaller fish left Tank A first, we did not find significant differences with regard to fish size.However, the control fish left Tank A in groups, as compared to the fish fed A. platensis who left individually, which supports the idea that when fish which feel threatened they seek the safety of others.Finally, although it was not our aim to measure resistance to hypoxia, the results also suggest that SPR fish were more tolerant to low oxygen levels.This is in accordance with other studies in humans, where Arthrospira sp.supplementation increases cardiovascular capacity (Kalafati et al., 2010).Moreover, it has been found that Arthrospira sp.increase hematocrit levels of O. niloticus (Ibrahem et al., 2013), which improves the efficiency of oxygen consumption.This finding is important for the aquaculture industry since oxygen levels are often a limiting factor for production (Abdel-Tawwab et al., 2015).
In summary, supplementing O. niloticus fry with A. platensis strain PCC 9108 increased survival and the proportion of individuals that adopted a proactive coping style, based on the results from a two-choice test.The results also suggest that A. platensis increased resistance to hypoxia, which is important for intensive aquaculture production.

Figure 1 .
Figure 1.Two-choice scheme with two tanks attached via a closable door.Tank A was in darkness and water was infused with nitrogen to decrease oxygen levels.Tank B was under natural light and had normal oxygen levels.Water flow in the tanks was opposite to avoid mixing water from one tank to another

Table 1 .
Summary of the four different solutions used to prepare the culture medium for Arthrospira platensis.

Table 2 .
Proximate composition of experimental diets.

Table 4 .
Mean exit times (±SD) of the first five fish during the two-choice test, mean oxygen levels when the first five fish left Tank A and the percentage of fish that left Tank A in groups.