Acessibilidade / Reportar erro

Effect of Inclusion of Arthrospira maxima Microalgae in Laying Hen Diets on Production Parameters and Egg Characteristics

ABSTRACT

The animal feed industry is continuously researching new feed additives to substitute other materials, reduce costs, or add value to the final product. The microalgae Arthrospira maxima, cultivated using wastewater as a nutritional source, was evaluated as a feed additive by including 2, 4, and 6% in isocaloric and isoprotein diets for laying hens. Five replicates per treatment with 5 hens per cage were used during an experimental period of 28 days. Productive behavior and egg characteristics (quality, fatty acid profile, cholesterol level) were evaluated. The inclusion of microalgae up to 4% in diets for 52-week-old laying hens did not affect productive performance (egg production, egg weight, egg mass, FCR, shell thickness, and Haugh units). No effects were observed on the cholesterol level or the concentration of fatty acids in the eggs, but more information is needed to determine if the microalgae drying or storage process can generate variations of these results. The inclusion of 6% microalgae produced an acceptable egg yolk color for the local market. The results indicate that this material can be used as a protein source up to an inclusion of 4% in the diet, and hens may need an adaptation period to maintain production at the 6% inclusion level.

Keywords:
Spirulina; poultry; feedstuff; Egg Quality; fatty acids

INTRODUCTION

The demand for soy and maize for animal feed production has accelerated in the last 5 years to support the increasing demand for protein sources for animal production. In 2021, 195.5 thousand metric tons of soy were produced in South America, with approximately 75% destined for animal feed (Cargill, 2021). The Chamber of Balanced Food Industrialists (CIAB by its Spanish acronym) showed in their annual report (2021) a production of 142,533 metric tons of poultry feed in 2020 to sustain the production of 3.55 million laying hens, mainly made from imported sources such as maize and soy. Therefore, there is a constant need to evaluate alternative feed sources to reduce dependence on these resources and add value to local production. A new trend has emerged for animal protein production using other feed and additives capable of being as efficient as antibiotics, adding pigments or other biomolecules (Mariey et al., 2012Mariey YA, Samak HR, Ibrahem MA. Effect of using Spirulina platensis algae as afeed additive for poultry diets: 1-productive and reproductive performances of local laying hens. Egyptian Poultry Science Journal 2012;32(1):201-15.), such as microalgae. In literature, the inclusion of the marine microalgae Dunaliella salina in the diets of laying hens improved performance, intestinal health, physicochemical quality of eggs, increased carotenoid content, and improved egg oxidative stability (Fernandes et al., 2020Fernandes RTV, Gonçalves AA, Martins AVA. Production, egg quality, and intestinal morphometry of laying hens fed marine microalga. Revista Brasileira de Zootecnia 2020;49:e20200011. https://doi.org/10.37496/rbz4920200011
https://doi.org/10.37496/rbz4920200011...
). In Japanese quails, birds fed with the microalgae Arthrospira platensis increased yolk index, color intensity, antioxidant capacity, and mono-unsaturated fatty acid levels of the yolks; decreased the levels of saturated and polyunsaturated fatty acids in egg yolks, and levels of lipid peroxidation were also lower (Boiago et al., 2019Boiago MM, Dilkin JD, Kolm MA, et al. Spirulina platensis in japanese quail feeding alters fatty acid profiles and improves egg quality: benefits to consumers. Journal of Food Biochemistry 2019;43(7):e12860. https://doi.org/10.1111/jfbc.12860
https://doi.org/10.1111/jfbc.12860...
).

Among the alternative microalgae species, Arthrospira maxima and A. platensis (commercially known as spirulina) can be produced autotrophically, heterotrophically in the absence of light with an organic carbon source (i.e., glycerol) and, mixotrophically (carbon and nutrients source derived from excreta and wastewater) (Ortiz-Moreno et al., 2012Ortiz-Moreno ML, Cortés-Castillo CE, Sánchez-Villarraga J, et al. Evaluación del crecimiento de la microalga Chlorella sorokiniana en diferentes medios de cultivo en condiciones autotróficas y mixotróficas. Orinoquia 2012;16(1):11-20.). The main advantage of mixotrophic microalgae cultivation is the reduction in production costs by up to 80% due to a decrease in inorganic nutrient supply in culture media. This suggests an advantage in industrializing cultivation as a component of circular economies in livestock production (i.e., pig or chicken farms) and obtaining nutrient-rich biomass as a food resource (Pereira et al., 2019Pereira MI, Chagas BM, Sassi R, et al. Mixotrophic cultivation of Spirulina platensis in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity. PloS One 2019;14(10):e0224294. https://doi.org/10.1371/journal.pone.0224294
https://doi.org/10.1371/journal.pone.022...
). A. maxima has been perceived as a feed supplement, as it has demonstrated beneficial functional properties in animal health, and its nutritional characteristics are complementary to soybean meal (Alvarenga et al., 2011Alvarenga RR, Borges Rodrigues P, Souza Cantarelli V de, et al. Energy values and chemical composition of spirulina (spirulina platensis) evaluated with broilers. Revista Brasileira de Zootecnia 2011;40(5):992-96. https://doi.org/10.1590/S1516-35982011000500008
https://doi.org/10.1590/S1516-3598201100...
).

Studies have reported on the palatability, toxicity, digestion, antioxidant action, pigmenting capacity, anticancer properties, immune stimulation, and anti-inflammatory capacity of these microalgae in rabbits, rats, chickens, and pigs (Belay et al., 1996Belay A, Kato T, Ota Y. Spirulina (Arthrospira): potential application as an animal feed supplement. Journal of Applied Phycology 1996;8(4-5):303-11. https://doi.org/10.1007/BF02178573
https://doi.org/10.1007/BF02178573...
; Grinstead et al., 2000Grinstead G.S, Tokach MD, Dritz SS, et al. Effects of spirulina platensis on growth performance of weanling pigs. Animal Feed Science and Technology 2000;83(3/4):237-47. https://doi.org/10.1016/S0377-8401(99)00130-3
https://doi.org/10.1016/S0377-8401(99)00...
; Rodrıguez-Hernández et al., 2001; Derner et al., 2006Derner, RB, Ohse S, Villela M, et al. Microalgas, produtos e aplicações. Ciência Rural 2006;36(6):1959-67. https://doi.org/10.1590/S0103-84782006000600050
https://doi.org/10.1590/S0103-8478200600...
; Colla et al., 2007Colla LM, Furlong EB, Costa JAV. Antioxidant properties of Spirulina (Arthospira) platensis cultivated under different temperatures and nitrogen regimes. Brazilian Archives of Biology and Technology 2007;50(1):161-7. https://doi.org/10.1590/S1516-89132007000100020
https://doi.org/10.1590/S1516-8913200700...
; Ambrosi et al., 2008Ambrosi MA, Costa JA, Bertolini T, et al. Propriedades de saudé de Spirulina spp. Revista de Ciências Farmacêuticas Básica e Aplicada 2008;29(2):109-17.; Peiretti & Meineri, 2011Peiretti PG, Meineri G. Effects of diets with increasing levels of Spirulina platensis on the carcass characteristics, meat quality and fatty acid composition of growing rabbits. Livestock Science 2011;140(1-3):218-24. https://doi.org/10.1016/j.livsci.2011.03.031
https://doi.org/10.1016/j.livsci.2011.03...
; Oliveira et al., 2013Oliveira CA de, Oliveira Campos AA de, Ribeiro SM, Castro Oliveira W de, Nascimento AG do. Potencial nutricional, funcional e terapêutico da cianobactéria spirulina. Revista da Associação Brasileira de Nutrição-RASBRAN 2013;19;5(1):52-9.; Moreira et al., 2015). All these properties are associated with the presence of biomolecules in Arthrospira maxima, such as phycocyanin, phenolic compounds, polyunsaturated fatty acids, carotenoids, and the antiviral calcium spirulan (Parra et al., 2017Parra J, Torres A, Rojas-Tortolero D, et al. Inclusión de la cianobacteria Arthrospira maxima como fuente de carotenoides en la dieta de gallinas ponedoras y su evaluación sobre la calidad del huevo. Revista Latinoamericana de Biotecnología Ambiental y Algal 2017;8(1):1-6.). Likewise, there are reports of this microalgae’s ability to color chicken egg yolks at the same level as commercial colorants without affecting the laying hens’ productive performance (Anderson et al., 1991Anderson DW, Chung-Shih T, Ernest R. The Xanthophylls of spirulina and their effect on egg yolk pigmentation. Poultry Science 1991;70(1):115-19. https://doi.org/10.3382/ps.0700115
https://doi.org/10.3382/ps.0700115...
; Zahroojian et al., 2011Zahroojian N, Moravej H, Shivazad M. Comparison of marine algae (Spirulina platensis) and synthetic pigment in enhancing egg yolk colour of laying hens. British Poultry Science 2011;52(5):584-88. https://doi.org/10.1080/00071668.2011.610779
https://doi.org/10.1080/00071668.2011.61...
; 2013)

In most of these studies, microalgae biomass was produced under autotrophic and heterotrophic conditions; however, little information is available on the effect of algae produced by mixotrophs as a feed additive on animal performance and diet cost. Recent studies have shown that mixotrophic cultivation of Arthrospira sp. in wastewater induces carbohydrate production and modification of its lipid profile (Hena et al., 2018Hena S, Znad H, Heong KT, et al. Dairy farm wastewater treatment and lipid accumulation by Arthrospira platensis. Water Research 2018;128:267-77. https://doi.org/10.1016/j.watres.2017.10.057
https://doi.org/10.1016/j.watres.2017.10...
; Pereira et al., 2019Pereira MI, Chagas BM, Sassi R, et al. Mixotrophic cultivation of Spirulina platensis in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity. PloS One 2019;14(10):e0224294. https://doi.org/10.1371/journal.pone.0224294
https://doi.org/10.1371/journal.pone.022...
). This study aimed to evaluate the inclusion of Arthrospira maxima obtained under mixotrophic conditions in diets for laying hens and its effect on productive performance and egg characteristics.

MATERIALS AND METHODS

The study was carried out at Los Pollitos poultry farm, located in the Zaragoza district of Alajuela province, Costa Rica, with minimum and maximum ambient temperatures of 17°C and 29°C (Costa Rican Meteorological Institute, 2019). A total of 100 ISA Brown laying hens, aged 52 weeks, were used. The laying hens were housed at room temperature, under a 16-hour lighting program, in battery cages with three levels (dimensions of the cage 30 x 30 x 80 cm), with 5 laying hens per cage (experimental unit), with 5 random replicates per treatment, for a total of 20 experimental units. The trial was approved by the Animal Use Ethics Committee of the University of Costa Rica (CICUA). The experimental period was 28 days and normal farm management was continued throughout the trial. The laying hens were fed daily, with a feed offer of 700 grams per cage at 6:00 am, and water was offered ad libitum through 2 nipples per cage. Feed residues (if any) were measured once a week for feed conversion ratio calculation, which considered the weekly mass of feed consumption per cage divided by the mass of eggs produced.

Microalgae biomass and diets

The biomass of the microalgae Arthrospira maxima was produced at the Biotechnology Research Center of the Costa Rica Institute of Technology (TEC, by its Spanish acronym). The culture medium was formulated from a wastewater dilution obtained from a pig farm, previously conditioned by anaerobic digestion for 27 days, and treated with sodium hypochlorite, which was later neutralized with sodium metabisulfite before inoculating the microalgae. The culture medium was supplemented with 10 g/L of sodium bicarbonate as a source of carbon and for pH regulation range (9-10). The initial inoculum was kept at 0.1 g/L of culture (17,000.00 cells/mL) and the chemical conditions of the culture medium were measured with QUANTOFIX® Relax plus kits (Machery-Nagel) and maintained over time at <200 mg/L SO42-, <4100 mg/L PO43-, <80 mg/L NH4+, <550 mg/L NO3-, and <400 mg/L K+. The microalgae culture was carried out in a continuous raceway system with a water level of 0.4 m deep and a volumetric capacity of 30,000 L. The culture was monitored for 35 days before downstream; cell growth was followed under an optical microscope (Leica DM 750) with a Neubauer cell counting chamber. Dry weight was measured using a halogen thermo-balance (Radwarg PNR 50). The ideal biomass density for harvesting was presented on day 35, reaching 0.71 g/L. A high culture yield of 0.98 g/L was obtained in the continuous phase while 30% of the culture was harvested weekly by centrifugation at 4200 rpm (Gea Westfallia SSD 606007 separator) and dried by spraying (Galaxie ECO Dryer® 1512) to obtain a fine green powder.

In vitro dry matter digestibility (Van Soest & Robertson 1979Van Soest P, Robertson J. Systems of analysis for evaluating fibrous feeds. Proceedings of the Workshop Standardization of Analytical Methodology for Feeds; 1979. Ottawa (CA): IDRC;1979.), microorganisms, and heavy metals in the biomass were analyzed as a qualitative assessment prior to designing feeding experiments.

Using the nutritional information of the algae (Table 1), four experimental diets were formulated using the Brill® software to meet the nutritional requirements of ISA Brown laying hens based on the Nutritional Guide for ISA Brown (Hendrix Genetics 2020b). The following microalgae nutrients were determined using AOAC methods (AOAC 2005) moisture 930.15, crude protein 990.0, crude fiber 962.09, ether extract 920.39, salt 969.10, ash 942.05, calcium 975.03, 968.08, phosphorus 965.17, 986.24, and sodium 985.35. Nitrogen-free extract was determined according to Morrison (1950Morrison FB. Feeds and feeding, a handbook for the student and stockman. 21st ed. Ithaca: The Morrison Publishing; 1950.) and total amino acids were determined according to Bartolomeo Maisano (2006Bartolomeo MP, Maisano F. Validation of a Reversed-Phase Liquid Chromatography Method for Quantitative Amino Acids Analysis. Journal of Biomolecular Techniques 2006; 17(2): 131-37.) and calculated as digestible according to Brazilian tables for birds and pigs, as well as metabolizable energy for birds (Rostagno et al., 2017). The diets were isoproteic (18% crude protein) and isoenergetic (2850 kcal/kg) with four levels of inclusion of Arthrospira maxima: T0 (0% microalgae - control), T2 (2% microalgae), T4 (4% microalgae), and T6 (6% microalgae). T0 maintained 3 mg/kg of canthaxanthin in the formulation due to normal market requirements for egg yolk color, while the other treatments did not have synthetic egg yolk pigment. Table 2 shows the composition of the diets.

Table 1
Nutritional composition of Arthrospira maxima biomass on dry mater basis.
Table 2
Ingredient composition of the experimental diets for laying hens, formulated using the software Brill® in as fed basis.1

Evaluated variables and sample collection

The productive performance of the hens was measured weekly, including feed consumption, egg weight, egg mass, and feed conversion rate (FCR, kg feed/kg eggs). In addition, eggs were graded, and the proportion of A-type eggs was calculated weekly using the following equation:

% T y p e A E g g s = ( T o t a l e g g s ( d i r t y e g g s + p a l e e g g + b r o k e n e g g s ) ) / T o t a l E g g s x 100

The egg quality was evaluated weekly using a sample of 15 eggs per treatment (3 eggs randomly selected from each replicate). Egg weight was determined using a precision balance (minimum detection of 0.1 g). Then, the egg content was placed on a flat surface, and the height of the dense albumen was measured using a caliper. Finally, the Haugh units were calculated using the following equation based on the egg weight (w, grams) and height of the dense albumen (h, millimeters):

U H = 100 x l o g [ ( h 1.7 x w x 0.37 + 7.6 ) ]

The yolk color was determined using the Digital Yolk Fan™ from DSM, and the eggshell thickness was measured using a digital caliper.

Additionally, nine eggs per treatment were randomly selected at the end of the experimental period to obtain a pooled sample for the analysis of fatty acid levels and cholesterol. The laboratory analysis was performed by the National Center for Food Science and Technology (CITA, for its acronym in Spanish) at the University of Costa Rica. The fatty acid profile was evaluated using AOAC 996.06 (AOAC 2005) and method Ce 1e-91, (AOCS, 2001) Gas Chromatography (GC-FID), P-SA-MQ-034. The cholesterol level was determined using high-pressure liquid chromatography (HPLC-PDA), developed according to the method described by Bauer et al. (2013Bauer LC, Santana DA, Macedo M, et al. Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD. Journal of the Brazilian Chemical Society 2013;161-68. https://doi.org/10.5935/0103-5053.20130283
https://doi.org/10.5935/0103-5053.201302...
).

Statistical Analysis

The variables were analyzed using statistical software R, version 3.5.1 (R Core Team 2018). Egg weight, egg mass, feed conversion ratio, shell thickness, and Haugh units were evaluated using mixed models of repeated measures, using the cage as the subject and the week as the time factor. Models for these variables with a normal distribution were adjusted using the complementary nlme package (Pinheiro et al., 2020Pinheiro J, Bates D, DebRoy S, et al. Nlme: linear and nonlinear mixed effects models (Version 3.1-147). 2020. Available from: https://CRAN.R-project.org/package=nlme.
https://CRAN.R-project.org/package=nlme...
). For A-type eggs, a beta distribution was assumed for the error, modeled with the complementary glmmTMB package (Brooks et al., 2017Brooks M, Kristensen K, Benthem K van, et al. glmmTMB. balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. The R Journal 2017;9:378-400. https://doi.org/10.32614/RJ-2017-066
https://doi.org/10.32614/RJ-2017-066...
). Finally, an ordinal logistic model was used for egg yolk color, adjusted with the ordinal package (Christensen, 2019Christensen, R. Ordinal: regression models for ordinal data. R Package Version 2019.12-10. Copenhagen; 2019.). After the described analysis, the Tukey test (p<0.05) was used to find differences between treatments. Since the concentration of egg fatty acids and cholesterol levels did not have a normal distribution, the variables were analyzed with a generalized beta linear model, with a logit function, using the betareg package (Cribari-Neto & Zeileis 2010Cribari-Neto F, Zeileis A. Beta regression in R. Journal of Statistical Software 2010;34:1-24. https://doi.org/10.18637/jss.v034.i02
https://doi.org/10.18637/jss.v034.i02...
).

Formulation Costs

The formulation cost of each experimental diet was determined using the value of each feed component and the estimated cost of microalgae production reported by TEC under experimental conditions.

RESULTS

Effect of Arthrospira maxima dietary inclusion on productive performance and egg composition

The result of the in vitro digestibility test for dry matter showed high digestibility (99.7 ± 0.01) g/100 g of dry matter with no microbial contamination. Table 3 shows the feed intake, and feed refusal was observed during the first week for laying hens fed the T 6% diet, probably due to lower palatability and adjustment to the new feed. The feed refusal did not show a negative effect on feed conversion ratio (FCR) and there was a tendency to increase feed conversion for the T6% treatment by 0.4 points with respect to the control at week 54, with no statistical difference (Figure 1, A).

Table 3
Feed intake (g/day) of laying hen fed with different inclusion of levels of Arthrospira maxima in the diets, during the experimental period (52 to 55 weeks of age).1

Figure 1
Effect of the inclusion of Arthrospira maxima in laying hen diets on percentage of (A) Feed refusal and (B) A-type eggs during the experimental period (52 to 55 weeks of age). (B) Different lower-case letters in the same week indicate significant differences between treatments (p<0.05) weekly. Different capital letters in the same treatment indicate significant differences between weeks in the same treatment (p<0.05). T 0% (Control), T2%, T 4% and T 6% (the percentage refers to the inclusion of A. maxima in the diet).

The inclusion of Arthrospira maxima in the diets did not affect weekly egg weight, egg mass production, or feed conversion ratio (FCR). Tables 4 to 6 show the absence of significant differences between treatments or a time effec0t for those variables. A significant difference was observed for the average egg mass production during the 4-week trial period (Table 5) due to a numerical difference in egg production for the T6 group during the first 2 weeks of the experiment that created a significant difference for the total egg mass.

Table 4
Effect of the inclusion of Arthrospira maxima in laying hen diets on the egg weight (g) throughout the experimental period. (52 to 55 weeks of age).1
Table 5
Effect of the inclusion of Arthrospira maxima in laying hen diets on the egg mass (kg/week) during the experimental period (52 to 55 weeks of age).1
Table 6
Effect of the inclusion of Arthrospira maxima in laying hen diets on feed conversion ratio (FCR, kg feed/kg eggs) during the experimental period (52 to 55 weeks of age).1,2

During the experimental period, a decrease in A-type eggs was observed for treatments T4 and T6 (p<0.05) (Figure 1, B). This was caused by an increase in dirty eggs for laying hens fed microalgae diets. The effect was due to green pigmentation of the eggshells caused by beak marks from laying hens after eating and drinking water, but diarrhea was not observed.

For egg composition, the results did not show differences in the concentration of total cholesterol levels due to the inclusion of A. maxima in the diets. The fatty acid profile indicated that the monounsaturated fatty acids (MUFA) were affected by the inclusion of A. maxima in the diets (p<0.05). T2 and T4 had the highest and lowest MUFA concentration per egg, respectively. The general results by treatment can be observed in Table 7, where an increase in fatty acids can be observed for T2, a decrease in T4, and again an increase for T6.

Table 7
Effect of the dietary inclusion of Arthrospira maxima in the fatty acid profile (g/100g) and cholesterol level1 (mg/100 g) of chicken eggs.1,2,3

Effect of the dietary inclusion of Arthrospira maxima on selected egg quality traits.

The eggshell thickness ranged between 0.38 and 0.45 mm across treatments and trial duration, and a small decrease during the last week of the trial cannot be attributed to microalgae, but it seems to be associated to the egg size, the possible causes of these results will be addressed in the discussion section. Furthermore, there was no effect on Haugh units due to the treatments, the results were 80 or more throughout the trial.

Effects on egg yolk color were observed in this trial (Table 8), where a higher inclusion of microalgae increased the color according to DSM color scale, and a time effect was also observed.

Table 8
Effect of the dietary inclusion of Arthrospira maxima in laying hen feed on egg yolk color (using a Digital Yolk Fan™ of DSM).

Finally, the cost of experimental diets was compared to the control treatment (T0). Adding Arthrospira maxima meal to laying hen diets under experimental conditions increased costs per 1000 kg of feed by US$3.72, US$7.59, and US$13.8 for T2, T4, and T6, respectively. This increase is due to the substitution of soybean meal (US$ 0.39/kg) with microalgae in the diet (US$ 0.79/kg). It was also observed that savings generated by eliminating the addition of canthaxanthin as an artificial pigment due to the incorporation of microalgae did not offset the costs compared to the control treatment (T0).

DISCUSSION

The qualitative analysis of A. maxima biomass demonstrated that it is safe for use as a feed additive when cultured in wastewater. As recently reported, Arthospira sp. tends to take up macronutrients and micronutrients from wastewater and has shown less absorption affinity for heavy metals (Cardoso et al., 2020). The digestibility test indicated high nutritional availability of the dry biomass, which complements the low crude fiber content, which usually is not metabolized. The microalgae culture production yield agreed with previous reports in raceway systems for Arthrospira sp. (Grahl et al., 2018Grahl S, Palanisamy M, Strack M, et al. Towards more sustainable meat alternatives: how technical parameters affect the sensory properties of extrusion products derived from soy and algae. Journal of Cleaner Production 2018;198:962-71.; Purohit et al., 2019Purohit A, Kumar V, Chownk M, et al. Processing-independent extracellular production of high purity C-phycocyanin from Spirulina platensis. ACS Biomaterials Science & Engineering 2019;12;5(7):3237-45. https://doi.org/10.1021/acsbiomaterials.9b00370.
https://doi.org/10.1021/acsbiomaterials....
). Nutrient and pH control in the medium allowed for continuous culture and extended logarithmic phase, avoiding the stationary phase of growth and ammonium toxicity that cause productivity reduction (Lu et al., 2019Lu Q, Han P, Chen F, et al. A novel approach of using zeolite for ammonium toxicity mitigation and value-added Spirulina cultivation in wastewater. Bioresource Technology 2019;280:127-35. https://doi.org/10.1016/j.biortech.2019.02.042.
https://doi.org/10.1016/j.biortech.2019....
).

Effect of Arthrospira maxima dietary inclusion on productive performance and egg composition

The egg production results of this trial are comparable to the ISA Brown standards for hen age (ranging from 91.3 to 89.8%; Hendrix-Genetics 2020a) and partially agree with Omri et al. (2019Omri B, Amraoui M, Tarek A, et al. Arthrospira platensis (Spirulina) supplementation on laying hens' performance: Eggs physical, chemical, and sensorial qualities. Foods. 2019;8(9):386. https://doi.org/10.3390/foods8090386
https://doi.org/10.3390/foods8090386...
), who determined that there is no significant difference in egg production when 1.5% and 2.5% Arthrospira maxima is added to the diets of 44-week-old Lohmann laying hens. They also did not find a time effect during the 6-week experimental period. In addition, Zahroojian, et al. (2013Zahroojian N, Moravej H, Shivazad M. Effects of dietary marine algae (Spirulina platensis) on egg quality and production performance of laying hens. Journal of Agricultural Science and Technology 2013;15:1353-60.) did not report any differences in the productive performance of 63-week-old Hy-line W36 laying hens that were supplemented with 1.5%, 2.0%, and 2.5% Arthrospira maxima during a 3.5-week experimental period. Our results indicate a lower overall egg mass for the T6 treatment group, which was likely affected by lower feed intake during the first two weeks of the trial. To our knowledge, there are no reports in the literature indicating a 6% Arthrospira sp. inclusion in the diet, and these results could indicate that laying hens need an adaptation period before they can reach their normal production performance.

On the other hand, different results are reported with laying hens where an increase of 4.99% in egg production was observed with the supplementation of 3% Arthrospira maxima in substitution of wheat bran during a 25-day trial (Parra et al., 2017Parra J, Torres A, Rojas-Tortolero D, et al. Inclusión de la cianobacteria Arthrospira maxima como fuente de carotenoides en la dieta de gallinas ponedoras y su evaluación sobre la calidad del huevo. Revista Latinoamericana de Biotecnología Ambiental y Algal 2017;8(1):1-6.). However, in that trial, the Arthrospira diet had 3.53% more protein and 8.59% more fat, giving the laying hens a nutritional advantage over the control.

Although there was a reduction in soybean meal in the diets, egg weight was not affected mainly due to the contributions of lysine and methionine from Arthrospira maxima, as reported previously (Alvarenga et al., 2011Alvarenga RR, Borges Rodrigues P, Souza Cantarelli V de, et al. Energy values and chemical composition of spirulina (spirulina platensis) evaluated with broilers. Revista Brasileira de Zootecnia 2011;40(5):992-96. https://doi.org/10.1590/S1516-35982011000500008
https://doi.org/10.1590/S1516-3598201100...
), both limiting amino acids for egg weight (Salvador & Guevara, 2013Salvador E, Guevara V. Desarrollo y validación de un modelo de predicción del requerimiento óptimo de aminoácidos esenciales y del comportamiento productivo en ponedoras comerciales. Revista de Investigaciones Veterinarias del Perú 2013;24(3):264-76.). Other results indicated an increase in egg weight of 0.42 g and 1.57 g supplemented with 1.5% and 2.5% A. platensis, respectively, becoming higher in crude protein content, compared to the control (Omri et al., 2019Omri B, Amraoui M, Tarek A, et al. Arthrospira platensis (Spirulina) supplementation on laying hens' performance: Eggs physical, chemical, and sensorial qualities. Foods. 2019;8(9):386. https://doi.org/10.3390/foods8090386
https://doi.org/10.3390/foods8090386...
).

Other authors found results that disagreed with the present study. Selim et al. (2018Selim S, Hussein E, Abou-Elkhair R. Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science 2018;82:1-3. https://doi.org// 10.1399/eps.2018.227
https://doi.org//...
) did not find any differences in egg production and egg mass in an 8-week supplementation trial with 38-46-week-old laying hens. However, during the last 4 weeks of their experiment, they observed an increase in both parameters, so the differences in the overall averages of our research could be due to their short experimental period and the fact that the laying hens need more time to adjust to diets with higher levels of inclusion to Arthrospira maxima. It is important to mention that the egg weight and egg mass obtained in this experiment for all treatments are higher than the ISA Brown standards for age (Hendrix-Genetics, 2020a). In Costa Rica, eggs are sold by the kilogram, so for producers, not only egg weight but also the total mass of eggs produced per batch is important.

A trend towards lower egg production was observed during the first and second week of the trial for T6, which could have been caused by a decrease in feed intake. The lower feed intake for T6 could be due to a lower palatability of the diet with a higher level of Arthrospira maxima. This behavior has not been reported in other literature for laying hens, however, as mentioned before, there are no other trials with such a high level of inclusion as 6%, as in the present study. Nguedia et al. (2019Nguedia G, Miégoué E, Tendonkeng F, et al. Effect of graded levels of spirulina (arthropsira platensis) on feed intake and in vivo digestibility of Trypsacum laxum in guinea pig (Cavia Porcellus L). Journal of Zoological Research 2019;13(1):20-31. https://doi.org/10.14302/issn.2694-2275.jzr-19-2695
https://doi.org/10.14302/issn.2694-2275....
) reported a decrease of 6.13 g/animal/day in dry matter intake for guinea pigs supplemented with 6% Arthrospira maxima compared to supplementation at 4%.

However, the lower feed intake did not result in a change in feed conversion ratio (FCR). The absence of differences in FCR indicates that the substitution of soybean meal as a protein source apparently did not affect the digestibility or utilization of nutrients due to its high biological value (Shimamatsu, 2004Shimamatsu, H. Mass production of Spirulina, an edible microalga. Hydrobiologia 2004;512:39-44. https://doi.org/10.1023/B:HYDR.0000020364.23796.04
https://doi.org/10.1023/B:HYDR.000002036...
; Alvarenga et al., 2011Alvarenga RR, Borges Rodrigues P, Souza Cantarelli V de, et al. Energy values and chemical composition of spirulina (spirulina platensis) evaluated with broilers. Revista Brasileira de Zootecnia 2011;40(5):992-96. https://doi.org/10.1590/S1516-35982011000500008
https://doi.org/10.1590/S1516-3598201100...
; Batista et al., 2013Batista, AP, Gouveia L, Narcisa M. et al. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Research 2013;2(2):164-73. https://doi.org/10.1016/j.algal.2013.01.004
https://doi.org/10.1016/j.algal.2013.01....
; Holman & Malau-Aduli, 2013Holman BW, Malau-Aduli AE. Spirulina as a livestock supplement and animal feed. Journal of Animal Physiology and Animal Nutrition 2013;97(4):615-23. https://doi.org/10.1111/j.1439-0396.2012.01328.x
https://doi.org/10.1111/j.1439-0396.2012...
; Sprujit et al., 2016Sprujit J, Van der Weide R, Van Krimpen M. Opportunities for micro algae as ingredient in animal diets. PPO 2016;712:1-46.). However, due to the nature of the commercial farm where this trial was conducted, there was no opportunity to weigh the laying hens during the experimental period.

The increase in second-grade eggs was due to the presence of dirtier eggs. The experimental diets turned green due to the inclusion of microalgae; the more microalgae included, the greener the mash became, caused by the dissolution of chlorophylls and phycocyanin highly concentrated in A. maxima. This caused a green liquid to drip from the beaks of the laying hens onto some of the eggs when they were laid. The green spotted eggs do not represent any risk for human consumption, due to the innocuity of the pigments.

The results of the total cholesterol level in this trial are contrary to the findings of Selim et al. (2018Selim S, Hussein E, Abou-Elkhair R. Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science 2018;82:1-3. https://doi.org// 10.1399/eps.2018.227
https://doi.org//...
) who reported that supplementation of 0.3% A. platensis led to a decrease in cholesterol content levels of eggs from 13.6 to 11.7 mg/g due to a decrease in serum triglyceride and cholesterol levels from 135 to 115 mg/dl. In addition, Mariey et al. (2012Mariey YA, Samak HR, Ibrahem MA. Effect of using Spirulina platensis algae as afeed additive for poultry diets: 1-productive and reproductive performances of local laying hens. Egyptian Poultry Science Journal 2012;32(1):201-15.) determined that supplementation of 0.2% A. platensis resulted in a reduction of 23.83 mg/100 ml of triglycerides, 7.34 mg/ml of plasma cholesterol level, 59.6 mg/g of total lipids, and 3.3 mg/g of cholesterol level in the egg yolk compared to a corn and soybean meal diet.

As shown in Table 7, only MUFA showed statistical differences. However, the differences do not show a trend according to the level of microalgae inclusion. This is due to the low ether extract (EE) content of the microalgae (Table 1), which does not have much participation in the fatty acid profile.

The deposition of fatty acids in the egg yolk is directly related to the content and composition of the ether extract (EE) of the diets offered to laying hens (Griffin & Hermier, 1988Griffin H, Hermier D. Plasma lipoprotein metabolism and fattening in poultry. In: Leclercq B, Whitehead CC, editors. Leanness in domestic birds. London:Butterworths, London; 1988. p.175-201. ISBN- 978-0408010368; Sartori et al., 2009Sartori ÉV, Canniatti-Brazaca SG, Cruz SH, et al. Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de postura e sua interferência na disponibilidade do ferro. Food Science and Technology. 2009;29:481-7. https://doi.org/10.1590/S0101-20612009000300004
https://doi.org/10.1590/S0101-2061200900...
; Cherian, 2015Cherian G. Nutrition and metabolism in poultry: role of lipids in early diet. Journal of Animal Science and Biotechnology 2015;6(1):28. https://doi.org/ 10.1186/s40104-015-0029-9.
https://doi.org/...
).

An increase in the yield of fatty acids in microalgae biomass produced in wastewater has been reported, which differs from the results of this study, where a lower lipid yield was obtained in relation to autotrophic cultures. The average fatty acid profile has been reported for this microalga, and it is mainly rich in palmitic acid and palmitoleic acid, each accounting for up to 40%, and oleic acid and linoleic acid with percentages ranging from 9 to 15%, as found in other feed matrices such as corn and soy. Other important lipids in A. maxima are omega-3 fatty acids such as linoleic acid and cis-8,11,14-eicosatrienoic acid and would represent a contribution to the fatty acid profile of the egg if the percentages in the biomass were higher than those found in the biomass used in this trial (Cañizares-Villanueva et al., 1995Cañizares-Villanueva RO, Domínguez AR, Cruz MS, et al. Chemical composition of cyanobacteria grown in diluted, aerated swine wastewater. Bioresource Technology 1995;51(2-3):111-16. https://doi.org/10.1016/0960-8524(94)00099-M
https://doi.org/10.1016/0960-8524(94)000...
; Borges et al., 2013Borges JA, Rosa GML, Meza LHR, et al. Spirulina Sp. LEB-18 culture using effluent from the anaerobic digestion. Brazilian Journal of Chemical Engineering 2013;30(2):277-88. https://doi.org/10.1590/S0104-66322013000200006
https://doi.org/10.1590/S0104-6632201300...
).

Finally, the drying method and storage time may have influenced the lipid profile of the microalgae meal. Long times of storage of microalgae biomass has shown oxidation of the fatty acids and a decrease in antioxidants. Nouri & Abbasi (2018Nouri E, Abbasi H. Effects of different processing methods on phytochemical compounds and antioxidant activity of Spirulina platensis. Applied Food Biotechnology 2018;5(4):221-32. https://doi.org/10.22037/afb.v5i4.20715
https://doi.org/10.22037/afb.v5i4.20715...
) found a 20% reduction in antioxidant activity in sun-dried Arthrospira maxima biomass compared to sealed vacuum oven drying. On the other hand, storage time and temperature have a negative effect on the antioxidant activity of Arthrospira maxima biomass (Colla et al., 2016Colla LM, Bertol CD, Ferreira DJ, et al. Thermal and photo-stability of the antioxidant potential of spirulina platensis powder. Brazilian Journal of Biology 2016;77(2):332-39. https://doi.org/10.1590/1519-6984.14315
https://doi.org/10.1590/1519-6984.14315...
). The inhibition on the functional effect expected for this trial, is attributed mainly in reducing cholesterol levels in eggs and increasing omega-6 fatty acids in eggs (Romay et al., 2003Romay CH, Gonzalez R, Ledon N, et al. C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Current Protein and Peptide Science 2003;4(3):207-16. https://doi.org/10.2174/1389203033487216
https://doi.org/10.2174/1389203033487216...
; Eriksen, 2008Eriksen NT. Production of Phycocyanin- a pigment with applications in biology, biotechnology, foods and medicine. Applied Microbiology and Biotechnology 2008;80(1):1-14. https://doi.org/10.1007/s00253-008-1542-
https://doi.org/10.1007/s00253-008-1542-...
; Mariey et al., 2012Mariey YA, Samak HR, Ibrahem MA. Effect of using Spirulina platensis algae as afeed additive for poultry diets: 1-productive and reproductive performances of local laying hens. Egyptian Poultry Science Journal 2012;32(1):201-15.; Cuellar‐Bermudez et al., 2015Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez DL, et al. Extraction and purification of high-value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins. Microbial Biotechnology 2015;8(2):190-209. https://doi.org/10.1111/1751-7915.12167.
https://doi.org/10.1111/1751-7915.12167...
; Selim et al., 2018Selim S, Hussein E, Abou-Elkhair R. Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science 2018;82:1-3. https://doi.org// 10.1399/eps.2018.227
https://doi.org//...
). In addition to lipid content, other important aspects of A. maxima cultivation, such as the ability to grow to reuse water, feasible biomass recovery from the culture medium, and high protein content, should be considered when choosing a microalga for animal feed.

Effect of the dietary inclusion of Arthrospira maxima on selected egg quality traits

The statistical analysis does not show a clear effect of treatments on shell thickness. However, a decrease can be observed for all treatments starting from the second week. Thinner eggshells may be related to the age of the hens, as it has been determined that eggshell quality decreases during the later egg production period (55 to 65 weeks) (Roberts et al., 2013Roberts JR, Chousalkar K. Egg quality and age of laying hens: implications for product safety. Animal Production Science 2013;53(12):1291-7. https://doi.org/10.1071/AN12345
https://doi.org/10.1071/AN12345...
). This phenomenon is related to the laying hen’s egg formation physiology, where the eggshell deposit remains the same throughout the production period, but the size of the egg increases over time, diluting the amount of shell and affecting its thickness. In addition, high ambient temperatures affect eggshell quality if hens begin to pant as a mechanism for evaporative cooling, causing an imbalance in blood pH that prevents calcium carbonate deposition in the eggshell (Allahverdi et al., 2013Allahverdi A, Feizi A, Takhtfooladi H, et al. Effects of heat stress on acid-base imbalance, plasma calcium concentration, egg production and egg quality in commercial layers. Global Veterinaria 2013;10:203-7. https://doi.org/10.5829/idosi.gv.2013.10.2.7286
https://doi.org/10.5829/idosi.gv.2013.10...
). Although temperature was not measured during this trial, the province of Alajuela has very hot days during the year, which could affect egg quality.

No effects were observed on the Haugh units in this trial due to the use of microalgae. It is important to mention that this quality indicator can be affected by other conditions such as laying hen age, feed formulation and management, environmental and egg storage temperature, egg age, and other non-dietary factors (Hy Line International, 2017).

Regarding egg yolk color, the addition of Arthrospira maxima affected the results, indicating that algae have pigmenting capacity despite a small numerical decrease in feed intake. However, a 6% inclusion of the microalgae was necessary to achieve the market color accepted by Costa Ricans (12 on the DSM scale) with a yolk color vary from yellow to orange, according to The Central American Technical Regulation RTCR 397:2006 (Ministerio de Agricultura y Ganadería, 2006). An effect due to time was also observed, in the case of the control diet (T0), there was a slight decrease in feed intake over the weeks, which could have affected pigment deposition. In the case of the experimental diets, it is necessary to investigate if the diets prior to the trial have a dragging effect on pigment deposition that dissipates over time. A longer observation period is recommended to evaluate if the color continues to decrease. Additionally, an effect of the drying process and storage time of the algae was addressed to explain the results in the fatty acid profile, and the same effects could be contributing to a lower pigmenting capacity of the microalgae due to oxidation.

These results are contrary to those reported by Zahroojian et al. (2013Zahroojian N, Moravej H, Shivazad M. Effects of dietary marine algae (Spirulina platensis) on egg quality and production performance of laying hens. Journal of Agricultural Science and Technology 2013;15:1353-60.), who determined that an inclusion of 2.5% freeze-dried Arthrospira maxima in diets without artificial colorants was sufficient to obtain a color score of 11.66 on the DSM scale, which means that a lower concentration of the microalgae was needed to achieve a high color score. These differences may be related to the effect of algae processing (freeze-drying extrusion) on the preservation of carotenoid pigments, where freeze-drying better preserves the components of microalgae and adds more color to the yolk (Anderson et al., 1991Anderson DW, Chung-Shih T, Ernest R. The Xanthophylls of spirulina and their effect on egg yolk pigmentation. Poultry Science 1991;70(1):115-19. https://doi.org/10.3382/ps.0700115
https://doi.org/10.3382/ps.0700115...
; Ross et al., 1994Ross E, Puapong DP, Cepeda FP, et al. Comparison of freeze-dried and extruded Spirulina platensis as yolk pigmenting agents. Poultry Science 1994;73(8):1282-9. https://doi.org/10.3382/ps.0731282
https://doi.org/10.3382/ps.0731282...
). In addition, it has been reported that the convection drying used to obtain the microalgae used in that trial decreases the effect of Arthrospira maxima on egg yolk pigmentation (Parra et al., 2017Parra J, Torres A, Rojas-Tortolero D, et al. Inclusión de la cianobacteria Arthrospira maxima como fuente de carotenoides en la dieta de gallinas ponedoras y su evaluación sobre la calidad del huevo. Revista Latinoamericana de Biotecnología Ambiental y Algal 2017;8(1):1-6.).

All treatments showed a decrease in egg yolk color in the time, which could indicate that there could be a residual effect of the control diet provided before the trial. A longer experimental period is needed to explore these results more deeply, but the results obtained were promissory.

In conclusion, the addition of Arthrospira maxima in diets of 52-week-old laying hens at levels of 2% and 4% did not affect the productive performance of the laying hens during a 4-week trial period (egg weight, egg mass, FCR, eggshell thickness, and Haugh units). The inclusion of microalgae at a level of 6% produced an acceptable egg yolk color on the DSM scale without artificial colorants in the feed, but a decrease in feed intake at the beginning of the trial created lower egg production and egg mass overall, suggesting an adaptation period for the hens to eat the higher inclusion level diet.

The use of Arthrospira maxima did not affect the cholesterol level or fatty acid concentration of the eggs. More information is needed to determine if the drying or storage process influenced these results.

The inclusion of Arthrospira maxima in diets increased the formulation costs under experimental conditions, so for the feed to be competitive in a lower cost feed formulation software, its cost should not be higher than the sum of the usual protein source plus the artificial colorant.

ACKNOWLEDGMENTS

The authors would like to thank Granja Avícola Los Pollitos for allowing the use of their chickens during this experiment, Vetim S.A. for mixing the diets used during the trial, and Luis Alejandro Rodríguez-Campos for his support in statistical analysis.

REFERENCES

  • Allahverdi A, Feizi A, Takhtfooladi H, et al. Effects of heat stress on acid-base imbalance, plasma calcium concentration, egg production and egg quality in commercial layers. Global Veterinaria 2013;10:203-7. https://doi.org/10.5829/idosi.gv.2013.10.2.7286
    » https://doi.org/10.5829/idosi.gv.2013.10.2.7286
  • Alvarenga RR, Borges Rodrigues P, de Souza Cantarelli V, Zangeronimo MG, da Silva Júnior JW, da Silva LR, Moreira dos Santos L, Pereira LJ. Energy Values and Chemical Composition of Spirulina (Spirulina Platensis) Evaluated with Broilers. Revista Brasileira de Zootecnia 2011; 40(5): 992-96.
  • Alvarenga RR, Borges Rodrigues P, Souza Cantarelli V de, et al. Energy values and chemical composition of spirulina (spirulina platensis) evaluated with broilers. Revista Brasileira de Zootecnia 2011;40(5):992-96. https://doi.org/10.1590/S1516-35982011000500008
    » https://doi.org/10.1590/S1516-35982011000500008
  • Ambrosi MA, Costa JA, Bertolini T, et al. Propriedades de saudé de Spirulina spp. Revista de Ciências Farmacêuticas Básica e Aplicada 2008;29(2):109-17.
  • Anderson DW, Chung-Shih T, Ernest R. The Xanthophylls of spirulina and their effect on egg yolk pigmentation. Poultry Science 1991;70(1):115-19. https://doi.org/10.3382/ps.0700115
    » https://doi.org/10.3382/ps.0700115
  • AOAC - Association of Official Analytical Chemists. Official methods of analysis. 18th ed. Gaithersburg; 2005. p.1-77.
  • AOCS - American Oil Chemists Society. AOCS surplus method Ce 1e-91. In: AOCS. Official methods and recommended practices. Illinois; 2001.
  • Bartolomeo MP, Maisano F. Validation of a reversed-phase liquid chromatography method for quantitative amino acids analysis. Journal of Biomolecular Techniques 2006;17(2):131-37.
  • Bartolomeo MP, Maisano F. Validation of a Reversed-Phase Liquid Chromatography Method for Quantitative Amino Acids Analysis. Journal of Biomolecular Techniques 2006; 17(2): 131-37.
  • Batista, AP, Gouveia L, Narcisa M. et al. Comparison of microalgal biomass profiles as novel functional ingredient for food products. Algal Research 2013;2(2):164-73. https://doi.org/10.1016/j.algal.2013.01.004
    » https://doi.org/10.1016/j.algal.2013.01.004
  • Bauer LC, Santana DA, Macedo M, et al. Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD. Journal of the Brazilian Chemical Society 2013;161-68. https://doi.org/10.5935/0103-5053.20130283
    » https://doi.org/10.5935/0103-5053.20130283
  • Belay A, Kato T, Ota Y. Spirulina (Arthrospira): potential application as an animal feed supplement. Journal of Applied Phycology 1996;8(4-5):303-11. https://doi.org/10.1007/BF02178573
    » https://doi.org/10.1007/BF02178573
  • Boiago MM, Dilkin JD, Kolm MA, et al. Spirulina platensis in japanese quail feeding alters fatty acid profiles and improves egg quality: benefits to consumers. Journal of Food Biochemistry 2019;43(7):e12860. https://doi.org/10.1111/jfbc.12860
    » https://doi.org/10.1111/jfbc.12860
  • Borges JA, Rosa GML, Meza LHR, et al. Spirulina Sp. LEB-18 culture using effluent from the anaerobic digestion. Brazilian Journal of Chemical Engineering 2013;30(2):277-88. https://doi.org/10.1590/S0104-66322013000200006
    » https://doi.org/10.1590/S0104-66322013000200006
  • Brooks M, Kristensen K, Benthem K van, et al. glmmTMB. balances speed and flexibility among packages for zero-inflated generalized linear mixed modeling. The R Journal 2017;9:378-400. https://doi.org/10.32614/RJ-2017-066
    » https://doi.org/10.32614/RJ-2017-066
  • Cañizares-Villanueva RO, Domínguez AR, Cruz MS, et al. Chemical composition of cyanobacteria grown in diluted, aerated swine wastewater. Bioresource Technology 1995;51(2-3):111-16. https://doi.org/10.1016/0960-8524(94)00099-M
    » https://doi.org/10.1016/0960-8524(94)00099-M
  • Cardoso Guimarães L, Hartwig Duarte J, Bomfim Andrade B, et al. Spirulina Sp. LEB 18 cultivation in outdoor pilot scale using aquaculture wastewater: high biomass, carotenoid, lipid and carbohydrate production. Aquaculture 2020;525:735272. https://doi.org/10.1016/j.aquaculture.2020.735272
    » https://doi.org/10.1016/j.aquaculture.2020.735272
  • Cargill. Soja de sudamérica: reporte de sostenibilidad de 2021. Actualización de mitad de año. Gargill; 2021. Available from: https://www.cargill.com/doc/1432194607736/soy-progress-mid-year-report-2021-es.pdf
    » https://www.cargill.com/doc/1432194607736/soy-progress-mid-year-report-2021-es.pdf
  • Cherian G. Nutrition and metabolism in poultry: role of lipids in early diet. Journal of Animal Science and Biotechnology 2015;6(1):28. https://doi.org/ 10.1186/s40104-015-0029-9.
    » https://doi.org/
  • Chiattoni LM, Machado MR, Rodrigues RD, et al. Influência do consumo de diferentes níveis de Spirulina no desenvolvimento e perfil lipídico de ratos. Revista Ceres 2015;62:142-8. https://doi.org/10.1590/0034-737X201562020003
    » https://doi.org/10.1590/0034-737X201562020003
  • Christensen, R. Ordinal: regression models for ordinal data. R Package Version 2019.12-10. Copenhagen; 2019.
  • CIAB. Informe Anual 2020: alimentos balanceados Costa Rica. San José: Cámara de Industriales de Alimentos Balanceados; 2020.
  • Colla LM, Furlong EB, Costa JAV. Antioxidant properties of Spirulina (Arthospira) platensis cultivated under different temperatures and nitrogen regimes. Brazilian Archives of Biology and Technology 2007;50(1):161-7. https://doi.org/10.1590/S1516-89132007000100020
    » https://doi.org/10.1590/S1516-89132007000100020
  • Colla LM, Bertol CD, Ferreira DJ, et al. Thermal and photo-stability of the antioxidant potential of spirulina platensis powder. Brazilian Journal of Biology 2016;77(2):332-39. https://doi.org/10.1590/1519-6984.14315
    » https://doi.org/10.1590/1519-6984.14315
  • Cribari-Neto F, Zeileis A. Beta regression in R. Journal of Statistical Software 2010;34:1-24. https://doi.org/10.18637/jss.v034.i02
    » https://doi.org/10.18637/jss.v034.i02
  • Cuellar-Bermudez SP, Aguilar-Hernandez I, Cardenas-Chavez DL, et al. Extraction and purification of high-value metabolites from microalgae: essential lipids, astaxanthin and phycobiliproteins. Microbial Biotechnology 2015;8(2):190-209. https://doi.org/10.1111/1751-7915.12167
    » https://doi.org/10.1111/1751-7915.12167
  • Derner, RB, Ohse S, Villela M, et al. Microalgas, produtos e aplicações. Ciência Rural 2006;36(6):1959-67. https://doi.org/10.1590/S0103-84782006000600050
    » https://doi.org/10.1590/S0103-84782006000600050
  • Eriksen NT. Production of Phycocyanin- a pigment with applications in biology, biotechnology, foods and medicine. Applied Microbiology and Biotechnology 2008;80(1):1-14. https://doi.org/10.1007/s00253-008-1542-
    » https://doi.org/10.1007/s00253-008-1542-
  • Fernandes RTV, Gonçalves AA, Martins AVA. Production, egg quality, and intestinal morphometry of laying hens fed marine microalga. Revista Brasileira de Zootecnia 2020;49:e20200011. https://doi.org/10.37496/rbz4920200011
    » https://doi.org/10.37496/rbz4920200011
  • Grahl S, Palanisamy M, Strack M, et al. Towards more sustainable meat alternatives: how technical parameters affect the sensory properties of extrusion products derived from soy and algae. Journal of Cleaner Production 2018;198:962-71.
  • Griffin H, Hermier D. Plasma lipoprotein metabolism and fattening in poultry. In: Leclercq B, Whitehead CC, editors. Leanness in domestic birds. London:Butterworths, London; 1988. p.175-201. ISBN- 978-0408010368
  • Grinstead G.S, Tokach MD, Dritz SS, et al. Effects of spirulina platensis on growth performance of weanling pigs. Animal Feed Science and Technology 2000;83(3/4):237-47. https://doi.org/10.1016/S0377-8401(99)00130-3
    » https://doi.org/10.1016/S0377-8401(99)00130-3
  • Hena S, Znad H, Heong KT, et al. Dairy farm wastewater treatment and lipid accumulation by Arthrospira platensis. Water Research 2018;128:267-77. https://doi.org/10.1016/j.watres.2017.10.057
    » https://doi.org/10.1016/j.watres.2017.10.057
  • Hendrix-Genetics. Brown. Cage production systems management report. Boxmeer; 2020a. Available from: https://www.isa-poultry.com/documents/598/ISA_Brown_CS_cage_English_management_report.pdf
    » https://www.isa-poultry.com/documents/598/ISA_Brown_CS_cage_English_management_report.pdf
  • Hendrix Genetics. Nutrition guide. Boxmeer; 2020b. Available from: https://layinghens.hendrix-genetics.com/documents/883/Nutrition_Guide_English_vs4.pdf
    » https://layinghens.hendrix-genetics.com/documents/883/Nutrition_Guide_English_vs4.pdf
  • Holman BW, Malau-Aduli AE. Spirulina as a livestock supplement and animal feed. Journal of Animal Physiology and Animal Nutrition 2013;97(4):615-23. https://doi.org/10.1111/j.1439-0396.2012.01328.x
    » https://doi.org/10.1111/j.1439-0396.2012.01328.x
  • Hy Line International. La Ciencia de la calidad del huevo [boletín técnico]. 2017. Available from: https://www.hyline.com/admin/Site/Controls/ViewFile?id=88df674d-2faf-411b-a883-d64770fcb740.
  • Instituto Meteorológico de Costa Rica. Meteorological data from the remote meteorological station of Alajuela Province, Grecia Canton, Argentina [personal communication]. Costa Rica: J Brenes; 2019.
  • Kumar KR, Mahadevaswamy M, Venkataraman LV. Storage quality of powdered cyanobacterium-spirulina platensis. Zeitschrift für Lebensmittel-Untersuchung und Forschung 1995;201:289-92. https://doi.org/10.1007/BF01193006
    » https://doi.org/10.1007/BF01193006
  • Lu Q, Han P, Chen F, et al. A novel approach of using zeolite for ammonium toxicity mitigation and value-added Spirulina cultivation in wastewater. Bioresource Technology 2019;280:127-35. https://doi.org/10.1016/j.biortech.2019.02.042
    » https://doi.org/10.1016/j.biortech.2019.02.042
  • Mariey YA, Samak HR, Ibrahem MA. Effect of using Spirulina platensis algae as afeed additive for poultry diets: 1-productive and reproductive performances of local laying hens. Egyptian Poultry Science Journal 2012;32(1):201-15.
  • Ministerio de Agricultura y Ganadería. Regulamento Técnico RTCR 397:2006 Huevos Frescos o Refrigerados de Gallina Para Consumo Humano. Costa Rica: Gobierno de la República; 2006.
  • Morrison FB. Feeds and feeding, a handbook for the student and stockman. 21st ed. Ithaca: The Morrison Publishing; 1950.
  • Nguedia G, Miégoué E, Tendonkeng F, et al. Effect of graded levels of spirulina (arthropsira platensis) on feed intake and in vivo digestibility of Trypsacum laxum in guinea pig (Cavia Porcellus L). Journal of Zoological Research 2019;13(1):20-31. https://doi.org/10.14302/issn.2694-2275.jzr-19-2695
    » https://doi.org/10.14302/issn.2694-2275.jzr-19-2695
  • Nouri E, Abbasi H. Effects of different processing methods on phytochemical compounds and antioxidant activity of Spirulina platensis. Applied Food Biotechnology 2018;5(4):221-32. https://doi.org/10.22037/afb.v5i4.20715
    » https://doi.org/10.22037/afb.v5i4.20715
  • Oliveira CA de, Oliveira Campos AA de, Ribeiro SM, Castro Oliveira W de, Nascimento AG do. Potencial nutricional, funcional e terapêutico da cianobactéria spirulina. Revista da Associação Brasileira de Nutrição-RASBRAN 2013;19;5(1):52-9.
  • Omri B, Amraoui M, Tarek A, et al. Arthrospira platensis (Spirulina) supplementation on laying hens' performance: Eggs physical, chemical, and sensorial qualities. Foods. 2019;8(9):386. https://doi.org/10.3390/foods8090386
    » https://doi.org/10.3390/foods8090386
  • Ortiz-Moreno ML, Cortés-Castillo CE, Sánchez-Villarraga J, et al. Evaluación del crecimiento de la microalga Chlorella sorokiniana en diferentes medios de cultivo en condiciones autotróficas y mixotróficas. Orinoquia 2012;16(1):11-20.
  • Parra J, Torres A, Rojas-Tortolero D, et al. Inclusión de la cianobacteria Arthrospira maxima como fuente de carotenoides en la dieta de gallinas ponedoras y su evaluación sobre la calidad del huevo. Revista Latinoamericana de Biotecnología Ambiental y Algal 2017;8(1):1-6.
  • Peiretti PG, Meineri G. Effects of diets with increasing levels of Spirulina platensis on the carcass characteristics, meat quality and fatty acid composition of growing rabbits. Livestock Science 2011;140(1-3):218-24. https://doi.org/10.1016/j.livsci.2011.03.031
    » https://doi.org/10.1016/j.livsci.2011.03.031
  • Pereira MI, Chagas BM, Sassi R, et al. Mixotrophic cultivation of Spirulina platensis in dairy wastewater: Effects on the production of biomass, biochemical composition and antioxidant capacity. PloS One 2019;14(10):e0224294. https://doi.org/10.1371/journal.pone.0224294
    » https://doi.org/10.1371/journal.pone.0224294
  • Pinheiro J, Bates D, DebRoy S, et al. Nlme: linear and nonlinear mixed effects models (Version 3.1-147). 2020. Available from: https://CRAN.R-project.org/package=nlme
    » https://CRAN.R-project.org/package=nlme
  • Purohit A, Kumar V, Chownk M, et al. Processing-independent extracellular production of high purity C-phycocyanin from Spirulina platensis. ACS Biomaterials Science & Engineering 2019;12;5(7):3237-45. https://doi.org/10.1021/acsbiomaterials.9b00370
    » https://doi.org/10.1021/acsbiomaterials.9b00370
  • R Core Team. R: a language and environment for statistical computing. R Foundation for Statistical Computing. Vienna; 2018. Available from: http://www.r-project.org/index.html
    » http://www.r-project.org/index.html
  • Roberts JR, Chousalkar K. Egg quality and age of laying hens: implications for product safety. Animal Production Science 2013;53(12):1291-7. https://doi.org/10.1071/AN12345
    » https://doi.org/10.1071/AN12345
  • Rodriguez-Hernández A, Ble-Castillo JL, Juarez-Oropeza MA, et al. Spirulina maxima prevents fatty liver formation in CD-1 male and female mice with experimental diabetes. Life Sciences 2001;69(9):1029-37. https://doi.org/10.1016/s0024-3205(01)01185-7
    » https://doi.org/10.1016/s0024-3205(01)01185-7
  • Romay CH, Gonzalez R, Ledon N, et al. C-phycocyanin: a biliprotein with antioxidant, anti-inflammatory and neuroprotective effects. Current Protein and Peptide Science 2003;4(3):207-16. https://doi.org/10.2174/1389203033487216
    » https://doi.org/10.2174/1389203033487216
  • Ross E, Puapong DP, Cepeda FP, et al. Comparison of freeze-dried and extruded Spirulina platensis as yolk pigmenting agents. Poultry Science 1994;73(8):1282-9. https://doi.org/10.3382/ps.0731282
    » https://doi.org/10.3382/ps.0731282
  • Rostagno HS, Albino LF, Donzele JL, et al. Tabelas brasileiras para aves e suínos. Composição de alimentos e exigências nutricionais. 2011;2:186. https://edisciplinas.usp.br/pluginfile.php/4532766/mod_resource/content/1/Rostagno%20et%20al%202017.pdf
    » https://edisciplinas.usp.br/pluginfile.php/4532766/mod_resource/content/1/Rostagno%20et%20al%202017.pdf
  • Salvador E, Guevara V. Desarrollo y validación de un modelo de predicción del requerimiento óptimo de aminoácidos esenciales y del comportamiento productivo en ponedoras comerciales. Revista de Investigaciones Veterinarias Del Perú 2013;24:264-76.
  • Salvador E, Guevara V. Desarrollo y validación de un modelo de predicción del requerimiento óptimo de aminoácidos esenciales y del comportamiento productivo en ponedoras comerciales. Revista de Investigaciones Veterinarias del Perú 2013;24(3):264-76.
  • Sartori EV, Canniatti-Brazaca SG, Cruz SH da, et al. Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de postura e sua interferência na disponibilidade do ferro. Ciência e Tecnologia de Alimentos 2009;29 (3):481-87. https://doi.org/10.1590/S0101-20612009000300004
    » https://doi.org/10.1590/S0101-20612009000300004
  • Sartori ÉV, Canniatti-Brazaca SG, Cruz SH, et al. Concentração de proteínas em gemas de ovos de poedeiras (Gallus gallus) nos diferentes ciclos de postura e sua interferência na disponibilidade do ferro. Food Science and Technology. 2009;29:481-7. https://doi.org/10.1590/S0101-20612009000300004
    » https://doi.org/10.1590/S0101-20612009000300004
  • Selim S, Hussein E, Abou-Elkhair R. Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science 2018;16(82):1-3. https://doi.org/10.1399/eps.2018.227
    » https://doi.org/10.1399/eps.2018.227
  • Selim S, Hussein E, Abou-Elkhair R. Effect of Spirulina platensis as a feed additive on laying performance, egg quality and hepatoprotective activity of laying hens. European Poultry Science 2018;82:1-3. https://doi.org// 10.1399/eps.2018.227
    » https://doi.org//
  • Shimamatsu, H. Mass production of Spirulina, an edible microalga. Hydrobiologia 2004;512:39-44. https://doi.org/10.1023/B:HYDR.0000020364.23796.04
    » https://doi.org/10.1023/B:HYDR.0000020364.23796.04
  • Sprujit J, Van der Weide R, Van Krimpen M. Opportunities for micro algae as ingredient in animal diets. PPO 2016;712:1-46.
  • Van Soest P, Robertson J. Systems of analysis for evaluating fibrous feeds. Proceedings of the Workshop Standardization of Analytical Methodology for Feeds; 1979. Ottawa (CA): IDRC;1979.
  • Zahroojian N, Moravej H, Shivazad M. Comparison of marine algae (Spirulina platensis) and synthetic pigment in enhancing egg yolk colour of laying hens. British Poultry Science 2011;52(5):584-88. https://doi.org/10.1080/00071668.2011.610779
    » https://doi.org/10.1080/00071668.2011.610779
  • Zahroojian N, Moravej H, Shivazad M. Effects of dietary marine algae (Spirulina platensis) on egg quality and production performance of laying hens. Journal of Agricultural Science and Technology 2013;15:1353-60.
  • FUNDING

    This research was funded by the Technological Institute of Costa Rica (TEC) through project No. 1510105 “Protein and lipid characterization of biomass obtained from microalgae Isochrysis galbana and Arthrospira maxima as a potential source of functional food” and the University of Costa Rica (UCR) through project No. 739-B8-209 “Use of microalgae in the feeding of poultry and pigs”.

Publication Dates

  • Publication in this collection
    20 Oct 2023
  • Date of issue
    2023

History

  • Received
    15 Dec 2022
  • Accepted
    11 July 2023
Fundação de Apoio à Ciência e Tecnologia Avicolas Rua Barão de Paranapanema, 146 - Sala 72, Bloco A, Bosque., CEP: 13026-010. , Tel.: +55 (19) 3255-8500 - Campinas - SP - Brazil
E-mail: revista@facta.org.br