Effect of dietary Spirulina (Arthrospira) platensis on the growth performance,

antioxidant enzyme activity, nutrient digestibility, cecal microflora, excreta

noxious gas emission, and breast meat quality of broiler chickens

J. H. Park, S. I. Lee, and I. H. Kim1

Department of Animal Resource and Science, Dankook University, Cheonan 31116, Republic of Korea

ABSTRACT This study examined the effects of di-
etary Spirulina (Arthrospira) platensis supplementation
on growth performance, antioxidant enzyme activity,
nutrient digestibility, cecal microflora, excreta noxious
gas emission, organ weight and breast meat quality in
broiler chickens. In total, 800 Ross 308 male broiler
chickens (1-d-old) were randomly divided into 5 dietary
treatments with 10 replicate cages (16 birds/replicate)
per treatment for 5 wk. The dietary treatments were
a control basal diet without Spirulina or with 0.25,
0.5, 0.75, or 1.0% Spirulina. Body weight gain, feed
conversion, and/or European production efficiency in-
dex improved linearly with supplementation of Spir-
ulina during d 8 to 21, 22 to 35, and overall d 1 to 35
(P < 0.05). Dietary Spirulina supplementation caused
a significant increase in the serum enzyme activity of
superoxide dismutase and glutathione peroxidase (lin-
Key words: ammonia gas emission, glutathione peroxidase, intestinal microorganism, superoxide dismutase,
drip loss
ear, P < 0.05). Apparent total tract digestibility of dry
matter and nitrogen showed a linear increase in Spir-
ulina supplementation (P < 0.05). Cecal Lactobacillus
count linearly increased and excreta ammonia gas emis-
sion linearly decreased, as dietary Spirulina supplemen-
tation increased (P < 0.05). There were no significant
effects on relative organ weight and breast meat qual-
ity of broilers fed with Spirulina diets; however, 7 d
drip loss linearly decreased in treatment groups fed
with Spirulina (P < 0.05). These results indicate that
adding Spirulina to the diet of broilers can improve an-
tioxidant enzyme activity, dry matter and nitrogen di-
gestibility, cecal Lactobacillus population, excreta am-
monia gas emission, and 7 d drip loss of breast meat.
In addition, dietary inclusion of 1.0% Spirulina pow-
der might provide a good alternative to improve broiler
chicken production.
2018 Poultry Science 97:2451–2459
http://dx.doi.org/10.3382/ps/pey093

INTRODUCTION Joventino et al., 2012). Many research studies have

Microalgae are attracting attention as the future
clean energy and industrial material resources such as
food, drug, cosmetics, and organic fertilizers because
they can be mass-produced in a short time in various
environments. In addition, Chlorella, Schizochytrium,
and Spirulina are recognized as renewable substitutes
for conventional protein sources (e.g., soybean meal,
fish meal, rice bran) in aquaculture or animal feed
because of their nutritional importance (Shields and
Lupatsch, 2012). Spirulina (Arthrospira) platensis is a
filamentous blue-green microalgae (cyanobacteria) gen-
erally regarded as a rich source of high quality pro-
tein, vitamins (particularly vitamin B12 and provitamin
β -carotene), minerals, essential fatty acids, essential
amino acids, pigments, and phenolic acids (Kul-
shreshtha et al., 2008; Bhavisha and Parula, 2010;

C 2018 Poultry Science Association Inc.
Received September 21, 2017.
Accepted March 7, 2018.
1
Corresponding author: inhokim@dankook.ac.kr
shown that Spirulina has antioxidant, immunomodu-
latory, anti-inflammatory, antiviral, and antimicrobial
activity in various experimental animals (Rasool et al.,
2009; Uyisenga et al., 2010; Langers et al., 2012; Abdel-
Daim et al., 2013; Shokri et al., 2014). Recently, there
has been a growing interest in its application in ani-
mals for its antioxidant activity, growth-promoting role,
and immunomodulatory effects. These positive effects
of Spirulina in the body may ultimately lead to im-
proved animal productivity. For example, in feeding tri-
als with livestock animals, Spirulina has been found to
increase growth rate, nutrient utilization, disease re-
sistance, egg quality, and carcass quality in poultry,
pigs, and rabbit (Al-Batshan et al., 2001; Meineri et al.,
2009; Sujatha and Narahari, 2011; Evans et al., 2015).
However, present knowledge of broiler chicken response
to dietary Spirulina supplementation is relatively un-
known.
The purpose of this study was to investigate the effect
of Spirulina microalgae as a feed ingredient source in
broiler chicken diets. Feeding experiments with broiler

2451
2452 PARK ET AL.

Table 1. Ingredient composition of experimental diets (as-fed basis).

Ingredients, % Phase 1 (d 1 to 7) Phase 2 (d 8 to 21) Phase 3 (d 22 to 35)

Corn 35.22 39.82 37.56

Soybean meal 37.26 32.06 28.64
Wheat 15.00 15.00 20.00
Animal fat 4.15 5.07 6.61
Corn gluten meal 3.78 3.86 3.24
Monodicalcium phosphate 1.22 1.08 0.90
Limestone 1.80 1.64 1.61
Salt 0.36 0.36 0.63
Choline-chloride 0.14 0.13 0.12
L-Lysine 0.39 0.37 0.36
DL-Methionine 0.27 0.24 0.24
L-Threonine 0.09 0.07 0.07
Vit. Mix1 0.12 0.11 0.10
Min. Mix2 0.10 0.10 0.10
Phytase 0.10 0.10 0.10
Calculated values
ME, kcal/kg 3,000 3,100 3,200
CP, % 23.00 22.00 20.00
Dig. Lys, % 1.32 1.10 1.00
Dig. Met, % 0.52 0.50 0.38
Dig. Met + Cys, % 1.05 0.90 0.72
Dig. Thr, % 0.94 0.80 0.74
Dig. Iso, % 0.95 0.80 0.73
Dig. Val, % 1.09 0.90 0.82
Dig. Arg, % 1.45 1.25 1.10
Ca, % 1.00 1.00 0.90
Total P, % 0.80 0.80 0.75
Available P, % 0.45 0.45 0.35
Na, % 0.20 0.20 0.15
Analyzed values
CP, % 23.87 21.87 20.28
Lys, % 1.42 1.27 1.17
Met, % 0.64 0.58 0.56
Ca, % 1.05 0.92 0.85
P, % 0.74 0.76 0.72
1
Provided per kg of complete diet: 11,025 IU of vitamin A; 1,103 IU of vitamin D3; 44 IU of vitamin
E; 4.4 mg of vitamin K; 8.3 mg of riboflavin; 50 mg of niacin; 4 mg of thiamine; 29 mg of d-pantothenic;
166 mg of choline; 33 μ g of vitamin B12.
2
Provided per kg of complete diet: 12 mg of Cu (as CuSO4 .5H2 O); 85 mg of Zn (as ZnSO4 ); 8 mg of
Mn (as MnO2 ); 0.28 mg of I (as KI); 0.15 mg of Se (as Na2 SeO3 .5H2 O).

chickens were conducted to assess nutritional physio-
logical properties, as well as to investigate effects on
growth performance.
MATERIALS AND METHODS
Experimental protocols describing the management
and care of animals were reviewed and approved by the
Animal Care and Use Committee of Dankook Univer-
sity (Approval No. DK-1–1642), Republic of Korea.
Animals and Housing
A total of 800 male broiler chickens (1-d-old, Ross
308) were obtained from a commercial hatchery. Broiler
chickens of similar body weight (41.5 ± 0.5 g) were ran-
domly distributed into 5 groups (160 birds in 10 cages
per treatment, 16 birds/cage). Broilers were housed in
a temperature-controlled room with 3 floors of stainless
steel battery cages (124 cm-width × 64 cm-length × 40
cm-height), which allowed free access to feed and wa-
ter during the experimental period. They were kept in
a room with controlled temperature and light regimen
of 22L:2D for the entire experimental period. The envi-
ronmental temperature was maintained at 33◦ C for the
first week and then gradually reduced to 20◦ C by the
fifth week. Relative humidity was gradually increased
from 60% (d 1 to 21) to 70% (d 22 to 35).
Diets
Broilers were fed a corn/soybean-based basal diet for
35 d divided in 3 phases: Phase 1 (d 1 to 7), Phase 2 (d
8 to 21), and Phase 3 (d 22 to 35) (Table 1). The exper-
imental diets, in mash form, were formulated to meet
and exceed the nutrients requirements of NRC (1994)
and Korean Feeding Standard for Poultry (2012). The
dietary treatments were a control basal diet without
Spirulina or with 0.25, 0.5, 0.75, or 1.0% Spirulina.
A commercially available freeze-dried Spirulina pow-
der was provided by a private company (NeoEnBiz Co.,
Bucheon, Republic of Korea) and supplemented to the
basal diet, at the expense of soybean meal. Table 2
shows the nutrient composition of the freeze-dried Spir-
ulina powder.
 EFFECTS OF SPIRULINA IN BROILERS 2453
Table 2. Nutrient composition of freeze- Apparent Total Tract Digestibility
dried Spirulina powder at −50◦ C.
To determine the apparent total tract digestibility,
Items %
0.2% chromic oxide was added to the experimental di-
Moisture 5.4 ets 4 d prior to the collection period. Excreta were col-
Protein 56.7 lected daily for the last 3 d of the experiment, and
Fiber 0.01
Ash 7.8 placed into a 60◦ C oven for 72 h. After drying, exc-
Amino acid reta were pulverized to pass through a 1-mm screen,
Aspartate 6.2 and dry matter and nitrogen in diets and excreta were
Threonine 3.2
Serine 3.3 analyzed (methods 934.01 and 968.06; AOAC, 2000).
Glutamate 8.6 Chromium concentration was determined by atomic ab-
Proline 2.4 sorption spectrophotometry (UV-1201, Shimadzu, Ky-
Glycine 3.1
Alanine 4.8 oto, Japan). The equation for calculating digestibility
Valine 3.3 was as follows: digestibility (%) = (1 – ((Nf × Cd)/(Nd
Methionine 1.2 × Cf))) × 100, where Nf = nutrient concentration in
Isoleucine 2.6
Leucine 5.2 feces (% DM), Nd = nutrient concentration in diet (%
Tyrosine 2.4 DM), Cf = chromium concentration in feces (% DM),
Phenylalanine 2.7 and Cd = chromium concentration in diet (% DM).
Lysine 3.1
Histidine 0.9
Arginine 3.7
Fatty acid
Lauric acid (C12:0) 0.4
Myristic acid (C14:0) 0.7
Palmitic acid (C16:0) 15.5 Cecal Microflora Population
Stearic acid (C18:0) 3.2
Arachidic acid (C20:0) 0.4 One gram of cecal sample was blended with 9 mL of
Behenic acid (C22:0) 0.4 sterile peptone water and mixed for 1 min on a vortex
Lignoceric acid (C24:0) 0.4
Palmitoleic acid (C16:1) 0.7 stirrer. Viable counts of bacteria in the cecal samples
Oleic acid (C18:1) 32.5 were conducted by plating serial 10-fold dilutions (10−1
Linoleic acid (C18:2n−6)
α -Linolenic acid (C18:3n−3)
22.7
21.4
to 10−8 ) onto Lactobacilli MRS agar (Difco Labora-
Gadoleic acid (C20:1) 0.6 tories, Detroit, MI, USA) plates and MacConkey agar
(Difco Laboratories, Detroit, MI, USA) plates to isolate
Lactobacillus spp. and coliform bacteria, respectively.
The lactobacilli agar plates were then incubated for
48 h at 37◦ C under anaerobic conditions. The Mac-
Growth Performance Conkey agar plates were incubated for 24 h at 37◦ C
Body weight (BW) and feed intake (FI) per cage under aerobic conditions. After the incubation periods,
were recorded on d 7, 21, and 35, and the feed con- colonies of the respective bacteria were counted and ex-
version ratio (FCR) was calculated based on feed in- pressed as the logarithm of colony-forming units per
take divided by body weight gain (BWG). Mortality gram (log10 CFU/g).
was recorded daily, and percentage mortality was calcu-
lated throughout the study. The European production
efficiency index (EPEI) was calculated with following
formula. EPEI = (BW/d × survival rate/FCR × 10).
Excreta Noxious Gas Emission
During the last 3 d of the experiment, fresh exc-
Antioxidant Enzyme Activity Analysis reta samples were collected from each replication for
At the end of the experiment (35 d), blood samples analyzing ammonia, hydrogen sulfide, and total mer-
were collected from the left wing vein into K3 EDTA captan. The excreta samples were kept in 3 L sealed
vacuum tubes (Becton Dickinson Vacutainer Systems, plastic containers for 5 d at room temperature (24◦ C).
Franklin Lakes, NJ), and stored at 4◦ C. For serum anal- After the fermentation period, a Gastec (model GV-
ysis, approximately 3 mL of blood samples were cen- 100) gas sampling pump was utilized for gas detec-
trifuged at 4,000 × g for 15 min at 4◦ C, after which the tion (Gastec Corp., Tokyo, Japan). Concentrations of
serum was separated. ammonia, hydrogen sulfide, and total mercaptan were
Antioxidant enzyme activities, superoxide dismutase measured within the scope of 5.0 to 100.0 (No. 3La,
(SOD), and glutathione peroxidase (GPx) in serum detector tube; Gastec Corp.), 2.0 to 20.0 (No. 4LK, de-
were measured using a commercial kit from Cayman tector tube; Gastec Corp.), and 0.5 to 120.0 (No.70 and
Chemical Company (Cayman Chemical Co., Ann Ar- 70-L, detector tubes; Gastec Corp.) ppm. The adhesive
bor, MI, USA), according to the manufacturer’s instruc- plaster was punctured, and 100 mL of headspace air
tions. was sampled at approximately 3 cm above the excreta.
2454 PARK ET AL.

Table 3. The effect of dietary Spirulina supplementation on growth performance in broilers.1

Spirulina, % P-value
Items 0 0.25 0.5 0.75 1.0 SEM6 Linear Quadratic Cubic Quartic

d 1 to 7
BWG2 , g 80 82 80 83 84 1.30 0.0736 0.4412 0.5805 0.2245
FI3 , g 105 105 104 104 103 1.66 0.3858 0.9489 0.8201 0.6992
FCR4 1.309 1.285 1.292 1.254 1.232 0.028 0.0426 0.6853 0.8665 0.5565
d 8 to 21
BWG, g 637 645 646 658 665 8.10 0.0117 0.7731 0.9536 0.6035
FI, g 997 999 1,003 1,013 994 9.29 0.8367 0.3088 0.2805 0.6226
FCR 1.568 1.549 1.556 1.539 1.496 0.020 0.0199 0.3344 0.4054 0.7668
d 22 to 35
BWG, g 1,011 1,033 1,035 1,046 1,052 10.70 0.0077 0.5776 0.6815 0.6497
FI, g 1,753 1,784 1,773 1,789 1,792 14.31 0.0759 0.6158 0.5242 0.3930
FCR 1.735 1.729 1.715 1.711 1.706 0.019 0.2216 0.8641 0.9064 0.8453
Overall
BWG, g 1,729 1,760 1,762 1,787 1,801 14.58 0.0003 0.8313 0.6945 0.4344
FI, g 2,856 2,888 2,880 2,906 2,889 18.37 0.1507 0.3573 0.9714 0.3424
FCR 1.653 1.642 1.636 1.626 1.605 0.012 0.0044 0.5902 0.6570 0.9959
Mortality 5.2 5.3 5.0 4.4 4.2 0.59 0.7974 0.7811 0.6878 0.5317
EPEI5 284.2 291.4 292.6 301.7 308.0 4.08 0.0004 0.6911 0.7997 0.4758
1
Each treatment mean represents 10 replicates (16 birds/replicate).
2
Body weight gain.
3
Feed intake.
4
Feed conversion ratio.
5
European production efficiency index.
6
Standard error of means.

Breast Meat Quality and Relative Organ Organ weights, breast meat, and abdominal fat were
Weight expressed as a percentage of live BW.

Color values of breast meat were measured in 3

replicates using a Minolta colorimeter (CR-300, Tokyo,
Japan) calibrated with a standard white plate and
recorded as L∗ , a∗ , and b∗ values for lightness, red-
ness, and yellowness, respectively. The pH values of raw
breast meat were measured using a pH meter (NWK
Binar pH, K-21, Landsberg, Germany) after blending
10 g of finely homogenized sample with 90 mL of
double-distilled water. To estimate the cooking loss, raw
meat samples were packed into Cryovac Cook-In Bags
after weighing, and cooked in a water bath at 100◦ C
for 30 min. Samples were cooled at room temperature
for 1 h and reweighed. Cooking loss was calculated as
the weight difference between the initial raw and fi-
nal cooked samples. Water-holding capacity (WHC)
was determined following the method of Kristensen and
Purslow et al. (2001). Five grams of meat sample was
heated to 70◦ C in a water bath for 30 min. Samples
were then cooled with ice and subsequently centrifuged
at 4◦ C at 1,000 × g for 10 min. WHC (%) was calcu-
lated as the ratio of weight loss of the sample during
centrifugation, to that of the original liquid. Drip loss
(%) was measured for 3 cm × 3 cm cuts of breast meat,
which were weighed, hung in a zipper bag, and stored
at 4◦ C. After storage, moisture on the surface of the
meat slices was carefully removed and weighed at d 1,
3, 5, and 7 after the sample was taken. The initial and
final weight of each sample was used to calculate drip
loss.
The liver, spleen, bursa of Fabricius, breast meat, ab-
dominal fat, and gizzard were removed and weighed.
Statistical Analysis
All data were statistically analyzed using the GLM
procedure in SAS program (SAS Institute Inc., Cary,
NC). Polynomial contrasts were used to determine lin-
ear, quadratic, cubic, and quartic effects of increasing
Spirulina levels on all measurements. Cage was used as
an experimental unit for growth performance, nutrient
digestibility, and excreta noxious gas. The individual
bird was used as the experimental unit for blood oxi-
dant enzyme, cecal microflora, and meat quality mea-
surements. Alpha was set at 0.05.
RESULTS
Growth Performance
Dietary Spirulina supplementation linearly increased
for BWG during d 8 to 21, 22 to 35, and overall d 1 to
35, as the inclusion rate increased from 0 to 1.0% (P
< 0.05) (Table 3). Increasing dietary supplementation
of Spirulina had a positive linear effect on the FCR
during d 8 to 21 and overall (P < 0.05). The EPEI
was linearly increased associated with the inclusion of
graded levels of Spirulina in the diets. No treatment
effects were observed on FI and mortality throughout
all the phases of feeding.
 EFFECTS OF SPIRULINA IN BROILERS 2455
Table 4. The effect of dietary Spirulina supplementation on blood SOD and GPx in broilers (d 35).1

Spirulina, % P-value
Items 0 0.25 0.5 0.75 1.0 SEM4 Linear Quadratic Cubic Quartic

SOD2 , U/mL 4.4 5.5 5.8 5.9 6.2 0.31 0.0016 0.1332 0.3568 0.9541
GPx3 , mU/mL 38.6 44.7 43.2 46.0 45.6 0.76 < 0.0001 0.0096 0.0794 0.0109
1
Each treatment mean represents 20 replicates (2 birds/replicate).
2
Superoxide dismutase.
3
Glutathione peroxidase.
4
Standard error of means.

Table 5. The effect of dietary Spirulina supplementation on apparent total tract nutrient digestibility in
broilers.1

Spirulina, % P-value
Items, % 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic

Dry matter 67.9 68.5 69.7 70.1 71.1 0.68 0.0325 0.7084 0.7160 0.9944
Nitrogen 66.1 66.6 67.0 67.9 68.7 0.78 0.0157 0.6724 0.9796 0.8838
1
Each treatment mean represents 10 replicates (16 birds/replicate).
2
Standard error of means.

Table 6. The effect of dietary Spirulina supplementation on cecal microflora in broilers (d 35).1

Spirulina, % P-value
2
Items, log10 cfu/g 0 0.25 0.5 0.75 1.0 SEM Linear Quadratic Cubic Quartic

Lactobacillus 7.39 7.45 7.50 7.63 7.78 0.10 0.0092 0.4988 0.9625 0.8822
Coliforms 5.05 5.03 5.02 5.02 4.99 0.03 0.1179 0.9852 0.7172 0.8297
1
Each treatment mean represents 20 replicates (2 birds/replicate).
2
Standard error of means.

Table 7. The effect of dietary Spirulina supplementation on excreta gas emission in broilers.1

Spirulina, % P-value
Items, ppm 0 0.25 0.5 0.75 1.0 SEM2 Linear Quadratic Cubic Quartic

Ammonia 45.1 35.9 34.1 31.0 31.2 0.26 0.0050 0.3213 0.6759 0.3082
Hydrogen sulfide 2.8 2.0 2.1 2.0 2.2 0.16 0.8497 0.4291 0.8869 0.5810
Mercaptan 3.3 2.5 2.1 2.4 2.8 0.77 0.9200 0.9729 0.9200 0.4987
1
Each treatment mean represents 10 replicates (16 birds/replicate).
2
Standard error of means.

Antioxidant Enzyme Activity icantly increased linearly as dietary Spirulina supple-

Antioxidant enzyme activity of Spirulina was evalu-
ated by analyzing serum SOD and GPx, and a linear
increase in these enzymes was observed with increasing
dietary levels of Spirulina (P < 0.0016 and P < 0.0001)
(Table 4).
Apparent Total Tract Digestibility
mentation increased (P < 0.05) (Table 6).
Excreta Noxious Gas Emissions
Excreta ammonia emissions decreased as dietary
Spirulina supplementation increased (linear, P < 0.05)
(Table 7). However, Spirulina supplementation did not
affect total mercaptan or hydrogen sulfide emissions.

The apparent total tract digestibility of dry matter

and nitrogen linearly increased in broiler chickens fed Meat Quality and Organ Weight
diets supplemented with 0 to 1.0% Spirulina (P < 0.05) There were no significant differences in pH, color (L∗ ,
(Table 5). a , b∗ ), cooking loss, or WHC of breast meat among
∗

the 5 treatment groups (P > 0.05) (Table 8). However,

Cecal Microbial Count
There was no significant difference in coliform bac-
teria counts of broiler chickens fed with different levels
of Spirulina. However, Lactobacillus counts were signif-
drip loss at 7 d post slaughter was significantly different
among the 5 groups. Birds fed with Spirulina showed
significantly lower drip loss as dietary levels of Spirulina
increased (linear, P < 0.05). Relative weights of most
organs (liver, spleen, gizzard, and bursa of Fabricius),
2456 PARK ET AL.
Table 8. The effect of dietary Spirulina supplementation on meat quality and relative organ weight in broilers
(d 35).1

Spirulina, % P-value
3
Items 0 0.25 0.5 0.75 1.0 SEM Linear Quadratic Cubic Quartic

pH value 5.74 5.89 5.85 6.15 6.15 0.17 0.0708 0.9484 0.8500 0.4222
Breast muscle color
Lightness (L∗ ) 41.60 45.49 45.14 43.18 46.47 1.34 0.1056 0.5878 0.0955 0.7116
Redness (a∗ ) 10.44 10.07 10.80 11.07 10.29 0.49 0.6645 0.5033 0.1920 0.8133
Yellowness (b∗ ) 9.33 9.64 9.93 8.46 9.86 0.77 0.9631 0.8878 0.2597 0.3436
Cooking loss 30.11 29.97 29.61 28.69 28.54 1.27 0.4882 0.9540 0.9484 0.9145
WHC2 , % 40.05 39.53 39.16 38.39 37.61 2.39 0.3575 0.6348 0.9257 0.9452
Drip loss, %
d1 5.73 5.59 5.54 5.51 5.36 0.72 0.7671 0.9667 0.8441 0.9246
d3 9.19 8.90 8.79 8.68 8.65 0.26 0.1472 0.6068 0.9082 0.9296
d5 11.34 10.92 10.86 10.82 10.76 0.28 0.1814 0.4833 0.6633 0.9004
d7 13.34 13.05 12.93 12.88 12.82 0.09 0.0009 0.1174 0.4892 0.9760
Relative organ weight, %
Breast muscle 18.55 19.10 19.13 19.61 19.73 0.87 0.3156 0.9020 0.9529 0.8111
Liver 2.86 2.80 3.07 2.88 2.77 1.34 0.8885 0.4932 0.7157 0.4625
Bursa of Fabricius 0.17 0.14 0.13 0.15 0.15 0.77 0.5623 0.1967 0.4832 0.5627
Abdominal fat 3.29 2.91 2.85 2.81 2.70 0.34 0.2624 0.6639 0.7225 0.9389
Spleen 0.13 0.11 0.10 0.11 0.11 0.01 0.3614 0.1943 0.6437 0.8215
Gizzard 1.30 1.18 1.14 1.16 1.13 0.07 0.1420 0.3963 0.5769 0.8689
1
Each treatment mean represents 20 replicates (2 birds/replicate).
2
Water holding capacity.
3
Standard error of means.

breast meat, and abdominal fat were not significantly
influenced by dietary supplementation of Spirulina.
DISCUSSION
This study found that broiler chickens fed diets
supplemented with Spirulina increased growth perfor-
mance. The mechanism of action of Spirulina has not
been clearly established, but previous studies have re-
ported that dietary supplementation with Spirulina
has positive effects on growth performance in poul-
try. Saxena et al. (1983) reported that White Leghorn
chicks fed experimental diets containing 111 g/kg and
166 g/kg Spirulina had greater weight gains at 6 wks
when Spirulina replaced groundnut cake. Venkatara-
man et al. (1994) reported that supplementation of
140 and 170 g/kg Spirulina, with no additional vita-
mins/minerals, could replace groundnut cake and fish-
meal with no adverse effects on broiler performance.
Raju et al. (2005) concluded that dietary inclusion of
Spirulina at 0.05% can partially alleviate adverse ef-
fects of 300 ppm aflatoxin on growth rate and lym-
phoid organ weight of broiler chickens. It has also been
reported that the amino acid pattern of Spirulina mi-
croalgae could be superior to the other vegetable feeds
(e.g., soybean meal), and that they have a high amino
acid digestibility (Alvarenga et al., 2011; Evans et al.,
2015). In addition, Spirulina contains physiologically
active substances such as carotenoid pigments, phyco-
cyanin, polyunsaturated fatty acid, vitamins, macro-
and micro-mineral elements, and many other chemical
compounds (Becker, 2007; Eriksen, 2008; Maoka, 2011).
These compounds confirm potential antimicrobial, an-
tioxidant, and anti-inflammatory biological properties,
or act as immune enhancers (Rathore et al., 2004;
Rasool et al., 2009; Uyisenga et al., 2010; Langers
et al., 2012; Abdel-Daim et al., 2013; Shokri et al.,
2014). Therefore, the chemical composition and phys-
iological functions of Spirulina seem to be involved
in metabolism systems related to growth performance,
and are likely to be the main cause of improvement of
BWG, FCR, and EPEI in broiler chickens.
GPx and SOD are generally thought to act as en-
zymatic free radical scavengers in cells (Abdel-Wahhab
and Aly, 2005). In this study, GPx and SOD linearly
increase in broiler chickens fed with Spirulina. Previous
studies indicated that Spirulina contains antioxidants
such as β -carotene, tocopherol, selenium, polypeptide
pigment, or phenolic acids, some of which might con-
tribute to antioxidant action together or with other
various micronutrients (El-Desoky et al., 2013). Specif-
ically, Spirulina is a rich source of phycocyanin, an
antioxidant biliprotein pigment, which is related to
other potent antioxidants (Khan et al., 2005). There
is almost no information on antioxidant properties re-
lated to Spirulina in poultry, but there is some evi-
dence of antioxidant activity from in vitro and several
rat studies. Estrada et al. (2001) suggested that pro-
tean extracts of Spirulina had scavenging effects against
hydroxyl radicals, with phycocyanin as the main com-
ponent responsible for the antioxidant activity. Addi-
tionally, β -carotene and other carotenoids protect cells
from oxidative stress by quenching singlet oxygen dam-
age through a variety of mechanisms (Tinkler et al.,
1994). Another probable cause is that increased levels
of blood SOD and GPx confirmed in Spirulina groups
may be associated with phenolic compounds. Many
 EFFECTS OF SPIRULINA IN BROILERS
phenolic compounds including salicylic, trans-cinnamic,
synaptic, chlorogenic, quinic, and caffeic acids present
in Spirulina may also be responsible for its antioxi-
dant activity, individually or synergistically (Miranda
et al., 1998). Wu et al. (2005) suggested that Spir-
ulina extract has stronger antioxidant capabilities than
Chlorella, which is probably due to higher content of
phenolic compounds (23.87 vs. 15.25 mg tannic acid
equivalent/g of algae aqueous extract) and antioxidant
capacity (ABTS assay: 19.74 vs. 4.60 μmol of Trolox
equivalent/g of microalgae). Therefore, increased serum
SOD and GPx concentrations in this study were likely
due to chemical compounds such as phycocyanin, β -
carotene, and phenolics in Spirulina, all relating to the
antioxidant activities.
In this study, the apparent total tract digestibilities
of dry matter and nitrogen linearly increased in broil-
ers fed with Spirulina diets, indicating that higher di-
gestibility could be achieved with higher concentrations
of Spirulina. The digestibility of Spirulina is not well
documented, and the available studies on assimilation
by poultry have not provided conclusive results. How-
ever, Mabeau and Fleurence (1993) confirmed that ma-
rine microalgae showed a high rate of protein degra-
dation proteolytic enzymes such as pepsin, pancreatin,
and pronase. Evans et al. (2015) reported that young
broilers (21-d-old) had higher apparent ileal digestibil-
ity of glutamic acid, proline, glycine, alanine, methion-
ine, leucine, and lysine when fed 6 to 21% Spirulina
supplemented diets compared with broilers fed con-
trol diets. Furbeyre et al. (2017) reported that the to-
tal tract digestibility in pigs receiving 1% Spirulina
and 1% Chlorella was greater for gross energy and
tended to be greater for dry matter, organic matter,
and neutral detergent fiber compared with control pigs.
They also found that villus height at the jejunum was
greater in pigs fed with Spirulina and Chlorella com-
pared with control pigs. Microalgae are generally re-
garded as a viable protein source, with essential amino
acid (EAA) composition meeting the Food and Agri-
culture Organization requirements, and are often on
par with other protein sources, such as soybean and
egg (Bleakley and Hayes, 2017). Increased digestibili-
ties of dry matter and nitrogen in this study may be
related to the high-quality protein containing balanced
EAA. Therefore, the better digestibility of protein ob-
served in Spirulina supplemented diets may be a result
of better absorption, which enhanced growth in broiler
chickens.
The cecum plays an important role in preventing
colonization of pathogens, detoxifying harmful sub-
stances, recycling nitrogen, microbial synthesis of vita-
mins, degradation of some carbohydrates, and absorb-
ing additional nutrients (Coates et al., 1968; Clench
and Mathias, 1995; Jorgensen et al., 1996). A previ-
ous study in broiler chickens also concluded that in-
testinal microbial-including cecum is highly associated
with the production performance of broiler chickens
(Jeong and Kim, 2014). Therefore, cecal microbial pop-
2457
ulations were investigated to evaluate Spirulina supple-
mentation in broiler chickens. This study indicated that
broiler chickens fed a Spirulina supplemented diet led
to higher cecal Lactobacillus concentration, but there
was no difference in the number of coliform bacte-
ria. Some studies suggest that microalgae have poten-
tial antibacterial, antiviral, and antifungal activities. In
vitro, de Mule et al. (1996) observed that methano-
lic and aqueous extracts of Spirulina inhibited the
growth of Candida albicans by 17.6%, whereas Lacto-
coccus lactis was promoted by the extract, with growth
increasing from 7.5 to 14.7%. Kaushik and Chauhan
(2008) demonstrated that the extracts of Spirulina have
shown antibacterial effects by inhibiting the growth
of harmful microorganisms, Staphylococcus aureus, Es-
cherichia coli, Pseudomonas aeruginosa, Salmonella ty-
phi, and Klebsiella pneumonia. The addition of dry
Spirulina at 10 mg/mL into de Man, Rogosa, and
Sharpe medium-promoted growth of Lactobacillus aci-
dophilus by 186%, suggesting prebiotic potential of the
microalgae (Bhowmik et al., 2009). Rania and Hala
(2008) also suggested that Spirulina extract had an-
tibacterial activities against E. coli because of the pres-
ence of alkaloids and lipopolysaccharides. In an in vivo
study, feeding Chlorella microalgae resulted in increased
Lactobacillus diversity in the crop or cecum or both of
laying hens (Janczyk et al., 2009; Kang et al., 2013). To
date, relatively few studies have investigated the an-
timicrobial activity of microalgae, including Spirulina
and their extracts in poultry. We hypothesized that
Spirulina supplementation would maintain the benefi-
cial microbial population, and subsequently explain the
improved digestibility and growth performance in this
study.
In regard to excreta noxious gas emission, the inclu-
sion of Spirulina linearly reduced the excreta ammo-
nia gas content as dietary levels of Spirulina increased.
Excreta noxious gas emission of animals is associated
with intestinal microflora, particularly harmful intesti-
nal bacteria populations (Ferket et al., 2002). A previ-
ous study also suggested that lower excreta noxious gas
content occurs when the microflora in the gastrointesti-
nal tract of broiler chickens is manipulated (Jeong and
Kim, 2014). Several other studies have also suggested
that excreta noxious gas content is associated with nu-
trient digestibility (Yan et al., 2011; Jeong and Kim,
2014), because the increased digestibility may allow less
substrate for microbial fermentation in the large in-
testine, which consequently decreases excreta noxious
gas content. In our study, the Spirulina supplemented
diet led to a better balanced microflora in the cecum
and higher nutrient digestibility than that by control
diet. Therefore, we suggest that the reason for reduc-
tion in excreta ammonia gas content may be the re-
sult of increased nitrogen digestibility and Lactobacillus
populations in broiler chicken ceca. There is currently a
lack of useful information regarding Spirulina in poul-
try, and previously reported functions of Spirulina in
other species cannot be directly compared. However,
2458 PARK ET AL.
findings from the current study support previous re-
search (Yan et al., 2012) showing that dietary Chlorella
supplementation decreased excreta ammonia gas emis-
sion in growing pigs.
Venkataraman et al. (1994) showed that color pig-
mentation of skin, breast, and thigh muscles was deeper
in broilers when substituting groundnut protein or fish
meal with Spirulina up to 170 or 140 g/kg. Toyomizu
et al. (2001) found that including Spirulina in broiler
feeds influenced both the yellowness and redness of
broiler meat. They reported that the increase of yel-
lowness with dietary Spirulina content may be reflected
in the common yellow pigment related to the accu-
mulation of zeaxanthin within the meat. However, un-
like previous reports that Spirulina has an effect on
meat color, this study showed no differences in breast
meat color of broiler chickens fed with Spirulina. Ad-
ditionally, there were no differences in pH, cooking
loss, WHC, or organ weights of broiler chickens fed
with Spirulina. Drip loss causes nutrients and moisture
to escape, and negatively affects chewiness of meat.
Spirulina-supplemented diets showed a significant re-
duction of drip loss after 7 d of storage. However, rea-
sons for this decrease remain unknown. Lu et al. (2014)
suggested that dietary addition of antioxidants was ef-
fective in improving growth, moderately restored whole
body antioxidant capability, and reduced drip loss. As-
ghar et al. (1989) and Attia et al. (2016) indicated
that antioxidants preserve the functionality of mem-
branes, thus improving their role as semipermeable bar-
riers against exudative loss. According to Cheah et al.
(1995), dietary antioxidant supplementation also pre-
vents excessive drip loss from pale, soft, exudative mus-
cle from stress-susceptible pigs. This action seems to
result from decreased membrane phospholipase activity
through higher antioxidant content of the tissue mem-
branes, because drip loss occurs when phospholipids in
the intracellular membrane are oxidized and the mem-
brane becomes weak. In this study, decreased drip loss
due to the inclusion of dietary Spirulina is, presum-
ably, related to the delayed oxidation of the cell mem-
brane. Improvement was probably due to the positive
effect of bioactive compounds, including antioxidants
of Spirulina, on the integrity of muscle fibers; thus, en-
hancing their capability to retain water (Dal Bosco et
al., 2014). The findings of our study support previous
research, which determined that Spirulina could affect
meat quality by decreasing the drip loss of Japanese
quail meats (Cheong et al., 2016). Further studies
are needed to determine the mechanisms involved
in the association between Spirulina and drip loss
of meat.
In conclusion, supplementation with Spirulina im-
proved BWG, FCR, and EPEI; increased antioxidant
enzyme activity, dry matter and nitrogen digestibili-
ties; increased cecal Lactobacillus populations; and de-
creased excreta ammonia gas emission in broiler chick-
ens. For breast meat quality, Spirulina decreased drip
loss after 7 d of storage. Therefore, Spirulina microal-
gae at 1.0% diet might provide a good alternative to
improve broiler chicken production.
ACKNOWLEDGMENTS
This research was financially supported by the Min-
istry of Trade, Industry, and Energy (MOTIE), Korea,
under the “Regional Specialized Industry Development
Program”(reference number R0005540) supervised by
the Korea Institute for Advancement of Technology
(KIAT).
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