Ingredients and feed preparation

To describe the contribution of spirulina (Spirulina maxima) in the vegetable protein diet, a feed based on wheat (Triticum sativum) and chickpea (Cicer arietinum) was prepared, containing 0% and 3% of spirulina. The wheat, chickpea and spirulina were obtained from local markets in Sinaloa, México. For the preparation of the feed, the powdered wheat and chickpea ingredients were mixed at a ratio of 2.5:1, adding the corresponding percentage of spirulina supplement (0% and 3% w/w), and mixed for 10 min, until their complete homogenization. The mixtures were adjusted to a moisture content of 12% (w/v) and were pelletized in a pelletizing machine (Model KL120B/C, China) with a production capacity of 70 to 100 kgh^-1. The vegetable mixtures were passed through a circular die with a diameter of 2.5 ' 3 mm, at a temperature of 60-80°C. The resulting pellets were dried in a stove (Felisa FE-132, México) under constant air flow at 85°C for 15 minutes. Then, the dried pellets were stored at room temperature (25°C). The formulation of the experimental feed is not included because it is in the process of patent registration (MX/E/2018/090734).

Proximal chemical analysis

The chemical analysis of the experimental feeds (0% and 3% of spirulina) were carried out using standard procedures of AOAC (^{AOAC, 1990}), for the determination of humidity by oven drying method (AOAC 930.15), ashes by calcination method (AOAC 942.05), protein by Kjeldahl method (AOAC 984.13), fats by the Soxhtlet method (AOAC 920.39), raw fiber by digestion method (AOAC 962.09) and carbohydrates by weight difference. The values were expressed as a percentage (%). Proximal chemical characterization was performed in triplicate for each feed.

Hydrostability

The percentage of hydrostability (%) of the experimental feeds was determined following the methodology described by ^{Obaldo et al., (2002)}. A sample of 25 g of each feed was homogenized in 100 mL of distilled water with continuous stirring at 200 rpm for 30 min. Subsequently, the mixture was filtered (850 urn), and the solid phase was dried at 130°C for 2 h. The percentage (% w/w) of hydrostability was determined from the weight of the resulting solid phase. The analysis was performed in triplicate.

Shrimp farming production

To carry out this study, four shrimp farms located in the Cospita (Farms A and B) and Elota (Farms C and D) areas of the State of Sinaloa participated consensually. Postlarvae of white shrimp (Litopenaeus vannamei) from the indicated farms were used. Prior to the start of the study, the health of the shrimp and the initial average weight of shrimps (0.17-0.2 g) were determined. In each participating farms (A-D), four stainless steel cages containing 10 postlarvae each per m² were placed: two cages that were randomly assigned for shrimp under the experimental feed and two cages for those under the control feed. The cages were lined with plastic mesh (1 m³) to isolate each experimental group. During the study, the saline concentration (mg-L^-1) of the water used for shrimp farming was measured as an environmental stability factor using a salinometer (Rhs-28ATC, YHequipment, China).

Growth and survival assessment

A field test was carried out for 5 weeks to evaluate the benefits of the experimental feed based on wheat and chickpea containing 3% of spirulina for the white shrimp growth in the pre-fattening phase. A commercial feed (MaltaCleyton®, Mexico) made from animal protein and other ingredients was used as a control. For the supply of feed, the initial average weight of ten shrimp per group was determined (2.56 ±1.2 g), which were placed in the cages and raised with the two different types of feed: control (commercial) and experimental (with Spirulina 3%). Feed was supplied every 12 h, and the daily feeding rate was slowly decreased from 10% to 8% of the shrimp biomass during the 35 days of the trial. Each week, all cages were collected to determine the weight and number of surviving shrimps of experimental group (n= 10). At the end of the experimental period, the shrimp cultured in the cages of all participating shrimp farms were harvested, counted, and weighed to determine the average weight gain (g), the biomass production (g), specific growth rate per day (g-day^-1) and survival rate (%). These variables were calculated with the following formulas:

Health analysis

For the health analysis, shrimp recovered after the raising period were sent to the State Committee of Aquaculture Health of Sinaloa (CESASIN). The health analysis included fresh bacteriological quantification and pathological study of hepatopancreas, gills, intestine and hemolymph of shrimp by histology. The microbiological analysis consisted of the quantification of bacteria (CFU/g) by shrimp organ. For the pathological analysis, the severity of the lesion was reported: negative or low (-), moderate (+) and high (++).

Feed characterization

The feed made from wheat and chickpea enriched with 3% spirulina, was characterized by a green color, cylindrical shape (2.5 mm in diameter) and smooth texture. The proximal chemical analysis of the experimental feeds (0% and 3% of spirulina) is shown in Table 1. The experimental feed with spirulina (3%) favors the protein content (17.5 ±0.1%) of the mixture of wheat and chickpea flours. Hydrostability in both experimental feeds was =58%.

Based on practice, it has been assumed that the way shrimp gain weight is directly related to the quality of the protein used in the diet⁹. Several sources of vegetable protein have been studied and recommended as an alternative for the total or partial replacement of protein of animal origin in food provided for the production of fish and crustaceans, due to the benefit reflected in their weight and size (^{Suárez et al., 2009}; ^{Ayisi et al., 2017}; ^{Cortés et al., 2021}). Additionally, corn, wheat and rice grains have been described as a source of carbohydrates that provide low-cost energy and improve pellet stability in water (^{Alhadi et al., 2013}) and the inclusion of legumes such as chickpea provide a higher bioavailable and digestible protein content (^{Reyes-Moreno et al., 2000}).

The commercial feed for shrimp farming has a higher protein content of animal origin (40%) than the experimental feed with 3% Spirulina maxima (17.5%). However, some authors have indicated that crude protein levels can be reduced without adverse effects on the performance variables of shrimp culture (^{Brito et al., 2018}; ^{Lee and Lee., 2018}). In this regard, ^{Lee and Lee (2018)}, observed that the performance of shrimp in the pre-fattening stage (0.65 g) did not vary when using diets with a crude protein content of 25 to 50%. ^{Velasco et al. (2000)}, reported that diets with a protein content of 18% provide a growth benefit in shrimp (L. vannamei) like the highest protein levels (25%) in the first stage of culture. Furthermore, ^{Lim and Persyn (1989)}, suggest that the protein value of diets of animal origin corresponds to crude protein that is not completely bioavailable. ^{Mente et al. (2002)}, describe that the total use of the protein depends on the constitution of the amino acids, the quality and digestibility. Therefore, the ingredients of the experimental diet with the inclusion of Spirulina (3%), had influential nutritional factors in the variables evaluated in the growth of shrimp in juvenile stage.

In this study, it was observed that the addition of Spirulina maxima improves the nutritional composition in comparison with the feed (chickpea and wheat) that does not contain it (Table 1). Recently, attention has been paid to the inclusion of chemical and natural attractors in diets to stimulate the intake and growth of farm animals (^{Ahamad et al., 2007}). ^{Jaime-Ceballos et al. (2017)}, pointed out the attraction power of the inclusion of Spirulina platensis to a commercial feed (animal protein) for shrimp farming. Therefore, it is possible that the inclusion of Spirulina maxima in the vegetable diet in this study may have presented a stimulating effect for the use of feed by shrimp.

Growth and survival assessment

Table 2 shows the growth and survival of shrimp cultured with the experimental and control diets.

The statistical analysis showed significant differences in final weight, average weight gain, biomass production and survival rate of the juvenile shrimp cultivated with the two types of feeds evaluated (P <0.05) (Table 2). It was determined that the shrimp fed with experimental diet presented a biomass production (0.74±0.17 g) and survival rate (>98.75%) better than shrimp fed with commercial diet (P <0.05). With both diets, an increase in the final weight of the shrimp was observed over time (r = 0.88, P <0.05) (Figure 1) and a similar daily growth rate of 0.22±0.05 g day^-1 (P > 0.05) (Table 2).

Figure 1 Daily growth rate of white shrimp (Litopenaeus vannamei) fed with the experimental feed based on wheat (Triticum sativum) and chickpea (Cicer arietinum) enriched with 3% spirulina (Spirulina maxima). The growth kinetics of the experimental group of shrimps for both foods was adjusted to a zero-order model (R² = 0.995), and the rate (μ) of total biomass (g-day^-1) during raising was not statistically significant (P >0.05). 

^{Suárez et al. (2009)}, have previously proposed the use of plant-based feeds as an alternative for shrimp farming since they favor feed conversion and the quality of the culture water. ^{Olmos et al. (2011)}, report having obtained a daily growth rate of biomass with the supply of a formulation of soy and Bacillus subtilis of 0.169 g-day^-1 and with a commercial food based on fish meal of 0.154 g-day^-1, during 20 days of shrimp culture in glass tanks. In the present study (Figure 1 and Table 2), it was possible to obtain a daily increase in biomass of 0.23 g-day^-1 in shrimp fed a diet of plant origin containing Spirulina, which is higher than previously reported. Likewise, the growth variables observed in this study agree with those previously reported for diets with protein content of 18% (^{Velasco et al., 2000}) and 25% (^{Lee and Lee, 2018}). Therefore, this study presents evidence of the feasibility of using a wheat and chickpea-based diet enriched with Spirulina maxima (3%), for feeding shrimp and potentially other crustaceans under farm conditions.

Currently, there is a trend to include immunostimulants into diets for disease prevention and reduction of early shrimp mortality (^{Wang et al., 2016}). ^{Gutierréz-Salmeán et al. (2015)}, published a review of the use of algae, in particular Spirulina, as a functional feed in diets for different experimental animals. They concluded that the inclusion of Spirulina up to a concentration of 5% in the diet did not cause any symptoms of toxicity. Additionally, ^{Nuhu (2013)}, reported that the addition of Spirulina maxima to the diet constitutes an ingredient that strengthens the immune system due to its nutritional, antioxidant, and antimicrobial properties. These reports are in accordance with the data in Tables 2 and 3 that indicate the superior survival and the best state of health of the shrimp fed with the vegetable feed, containing Spirulina maxima. Furthermore, ^{Chen et al. (2016)}, have shown the effect of the immunostimulant and preventive power of V. alginolyticus infection in shrimp fed with diets supplemented with Spirulina.

Health analysis

The health analysis of the shrimp fed with both feeds is shown in Table 3. In the fresh analysis of the hemolymph and the hepatopancreas, no filamentous bacteria were observed in the shrimp fed with the experimental and control feeds. The health variables of the gills and the hepatopancreas of both experimental groups were mostly at a low severity level. However, in the intestine, the low presence of gametocytes is shown in most of the shrimp fed with the experimental feed (60%) and the control feed (40%).

The health analysis shows that most of the analyzed shrimp present an acceptable health (Table 3). However, some specimens analyzed indicate damage to the hepatopancreas, gills and intestine. The development of these pathological damages has been related to stress and shrimp culture conditions (^{Borges et al., 2012}). The damage and lipid content in the hepatopancreas are related to its ability to metabolize nutrients, leading to altering the development of the crustacean (^{Gómez-Gil et al., 2001}). In this sense, it is observed that the shrimp of both experimental groups have a favorable response for the absorption of the feeds (Table 3). The presence of gametocytes is relevant given that their accumulation in the intestine of shrimp can favor the development of parasitic diseases (^{Fajer-Avila et al., 2005}). In this sense, it can be hypothesized that the bioactive properties of Spirulina (^{Nuhu, 2013}; ^{Gutiérrez-Salméan et al., 2015}; ^{Chen et al., 2016}) contained in the experimental feed reinforce the immune system of the crustacean during the stage of pre-fattening by favoring their survival in this study (Table 2).

Salinity monitoring in farm water

The salinity values of the farms are shown in Figure 2. Salinity is not related to survival (r = -0.156, P > 0.05) and daily weight (r = -0.004, P> 0.05) of the shrimp cultured with both feeds.

Figure 2 Saline concentration of water of shrimp's farms. The total daily weight of shrimp fed with the experimental feed with 3% Spirulina and commercial feed is represented by the black and gray spots, respectively. Values with different lowercase letters into parentheses are statistically different between farms (P <0.05). 

A physicochemical parameter that can condition shrimp growth and survival is the saline concentration of the culture water⁸. In addition, ^{Valenzuela-Quiñonez et al. (2010)}, indicates that shrimp specific growth can be observed with low salinity concentrations (1 mg-L^-1) and increased with values up to 35 mg-L^-1. Recognizing the indicated importance of salinity in the culture medium, the salinity values quantified in this study were within the marked limits for the water used in shrimp culture, according to the domestic criteria (27-35.0 mg-L^-1) of CE-CCA-001/89 (^{DOF, 1989}). The results of growth variables obtained with both feeding strategies (Figure 2), indicate that the salt concentration maintained the stability of these important quality variables. Moreover, these results indicate that the experimental feed used can be implemented in farms whose culture conditions are similar.