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Avian Pathology
ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/cavp20
Bioefficacy of dietary inclusion of Nannochloropsis
oculata on Eimeria spp. challenged chicks: clinical
approaches, meat quality, and molecular docking
Marwa I. Abdel Haleem, Hanem F. Khater, Shimaa N. Edris, Hanan A.A. Taie,
Samah M. Abdel Gawad, Nibal A. Hassan, Ali H. El-Far, Yasmeen Magdy &
Sawsan S. Elbasuni
To cite this article: Marwa I. Abdel Haleem, Hanem F. Khater, Shimaa N. Edris, Hanan A.A. Taie,
Samah M. Abdel Gawad, Nibal A. Hassan, Ali H. El-Far, Yasmeen Magdy & Sawsan S. Elbasuni
(19 Mar 2024): Bioefficacy of dietary inclusion of Nannochloropsis oculata on Eimeria spp.
challenged chicks: clinical approaches, meat quality, and molecular docking, Avian Pathology,
DOI: 10.1080/03079457.2024.2312133
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ORIGINAL ARTICLE
Bioecacy of dietary inclusion of Nannochloropsis oculata on Eimeria spp.
challenged chicks: clinical approaches, meat quality, and molecular docking
Marwa I. Abdel Haleem
a
, Hanem F. Khater
b
, Shimaa N. Edris
c
, Hanan A.A. Taie
d
,
Samah M. Abdel Gawad
b
, Nibal A. Hassan
e,f
, Ali H. El-Far
g
, Yasmeen Magdy
h
and Sawsan S. Elbasuni
a
a
Department of Avian and Rabbit Diseases, Faculty of Veterinary Medicine, Benha University, Benha, Egypt;
b
Department of Parasitology,
Faculty of Veterinary Medicine, Benha University, Benha, Egypt;
c
Department of Food Hygiene and Control, Faculty of Veterinary
Medicine, Benha University, Benha, Egypt;
d
Plant Biochemistry Department, National Research Centre, Dokki, Egypt;
e
Department of
Biology, Animal Reproduction Research Institute, Pathology Department, Giza, Egypt;
f
College of Science, Taif University, Taif, Saudi
Arabia;
g
Department of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt;
h
Department of
Anatomy and Embryology, Faculty of Veterinary Medicine, Benha University, Benha, Egypt
ABSTRACT
Although anticoccidial drugs have been used to treat avian coccidiosis for nearly a century,
resistance, bird harm, and food residues have caused health concerns. Thus, Nannochloropsis
oculata was investigated as a possible coccidiosis treatment for broilers. A total of 150
1-day-old male Cobb broiler chicks were treated as follows: G1-Ng: fed a basal diet; G2-Ps:
challenged with Eimeria spp. oocysts and fed basal diet; G3-Clo: challenged and fed basal
diet with clopidol; G4-NOa: challenged and fed 0.1% N. oculata in diet, and G5-NOb:
challenged and fed 0.2% N. oculata. Compared to G2-Ps, N. oculata in the diet significantly
(P < 0.05) decreased dropping scores, lesion scores, and oocyst shedding. Without aecting
breast meat colour metrics, N. oculata improved meat quality characters. At 28 days of age,
birds received 0.2% N. oculata had significantly (P < 0.05) higher serum levels of MDA, T-
SOD, HDL, and LDL cholesterol compared to G2-Ps. Serum AST, ALT, and urea levels were all
decreased when N. oculata (0.2%) was used as opposed to G2-Ps. Histopathological
alterations and the number of developmental and degenerative stages of Eimeria spp. in the
intestinal epithelium were dramatically reduced by 0.2% N. oculata compared to G2-Ps.
Molecular docking revealed a higher binding anity of N. oculata for E. tenella aldolase,
EtAMA1, and EtMIC3, which hindered glucose metabolism, host cell adhesion, and invasion
of Eimeria. Finally, N. oculata (0.2%) can be used in broiler diets to mitigate the deleterious
eects of coccidiosis.
ARTICLE HISTORY
Received 18 August 2023
Revised 3 January 2024
Accepted 20 January 2024
KEYWORDS
Broilers; marine microalgae;
lesion scoring; TEM;
oxidative stress biomarkers;
biochemical parameters
Introduction
The amount of poultry meat produced over the next
10 years is expected to equal half of global bird protein
production to fill the food gap caused by expanding
populations (de Mesquita Souza Saraiva et al., 2022).
Poor practices of some poultry farmers have impaired
immune function and spread disease, slowing devel-
opment and causing substantial economic losses
worldwide. Avian coccidiosis is a highly pathogenic
and contagious protozoan disease spreading mainly
in hot and humid environments (Moryani et al.,
2021). Seven highly host- and site-specific Eimeria
species infecting domestic chickens (Eimeria acervu-
lina, Eimeria brunetti, Eimeria maxima, Eimeria
mitis, Eimeria necatrix, Eimeria praecox, and Eimeria
tenella) are recognized as globally ubiquitous (Reid
et al., 2014; Saeed & Alkheraije, 2023). Birds become
infected after ingestion of sporulated oocysts. Clinical
signs of the disease include dysentery, bloody diar-
rhoea, enteritis, poor growth, drooped wings, emacia-
tion, and decreased production (Habibi et al., 2016;
Saeed & Alkheraije, 2023). Furthermore, coccidiosis
can result in concurrent infection with bacterial, fun-
gal, and viral pathogens, putting chicken ocks at risk
of morbidity and mortality (Moryani et al., 2021).
Coccidiosis can be successfully controlled by the
use of vaccinations and anticoccidial medications
(Kadykalo et al., 2018; Akanbi & Taiwo, 2020), such
as amprolium, clopidol, or halofuginone, which
directly aect the metabolism of coccidian species
(Kalkal et al., 2021). The extensive use and abuse of
medications over the past 40 years has caused various
detrimental impacts, including development of drug
resistance (Li et al., 2005; Zaheer et al., 2022), toxic
eects on bird byproducts when given without consid-
ering the suitable duration or dose (Sundar et al.,
2017), and food residues harming human health
(Mund et al., 2017).
Consumers and poultry farmers are currently seek-
ing natural, safe, and environmentally friendly pro-
ducts for treating coccidiosis in broiler chickens
(Abdel Haleem, Abdelnaser et al., 2019; Adjei-Mensah
& Atuahene, 2023; Ali et al., 2014, 2015) due to their
ability to enhance well-being, reduce the likelihood
© 2024 Houghton Trust Ltd
CONTACT Marwa I. Abdel Haleem marrwa.mahmoud@fvtm.bu.edu.eg
AVIAN PATHOLOGY
https://doi.org/10.1080/03079457.2024.2312133
of specific chronic illnesses, and provide health
advantages that extend beyond nutritional value
(Durmaz, 2007). Many natural materials, such as her-
bal extracts, essential oils, symbiotic, and organic
acids, have proven their eectiveness as alternatives
to chemical anticoccidial drugs in preventing cocci-
diosis. Microalgae are single-celled, eukaryotic, or
prokaryotic primary microorganisms with photosyn-
thetic activity that are used as natural feed additives
(Paterson et al., 2023). They contain a variety of
amino acids, essential fatty acids, carbohydrates,
and lipids, as well as microelements such as minerals,
vitamins, polyphenols, avonoids, carotenoids, and
natural antioxidant compounds (Saadaoui et al.,
2021; El-Sayed et al., 2022). Algae could exert protec-
tive eects against Eimeria challenge as a result of
improving intestinal integrity and systemic immune
responses (Fries-Craft et al., 2021). Microalgae of
the genus Nannochloropsis have cells ranging in size
from 2–4 µm to 3–41.5 µm (Al-Hoqani et al., 2017)
and were initially cut o from the coast of Scotland
(Ribeiro et al., 2020). There are currently seven
known Nannochloropsis species, six of which have
marine habitats (such as Nannochloropsis oculata),
and one of which is found in both fresh and brackish
waters (Al-Hoqani et al., 2017). Several studies have
shown that Nannochloropsis oculata (N. oculata)
has beneficial eects on the palatability and digestion
of food; moreover, it has detoxifying, antioxidant,
anti-inammatory, anticancer, and immune stimu-
lant properties (Derner et al., 2006; Colla et al.,
2007). N. oculata also improved growth, nonspecific
immunity, intestinal histomorphometry and resist-
ance to bacterial pathogens and upregulated interleu-
kin-10 gene expression after being added to the diet
of marine animals at a rate of 5% or 8% (Md et al.,
2018; Abdelghany et al., 2020). Additionally,
N. oculata is an excellent alternative to conventional
protein and eicosapentaenoic acid in animals diets
(Lacaz-Ruiz, 2003; Becker, 2007; Abd El-Hack et al.,
2023)
Accordingly, this study investigated the potential
benefits of incorporating N. oculata into chicken
feed as an alternative to anticoccidial medication,
and its ecacy on the clinical picture, performance,
meat quality, serum biochemistry, and intestinal histo-
morphometry of broiler chicks during coccidiosis
challenge.
Materials and methods
Ethical statement
All procedures involving birds complied with the
guidelines established by the Animal Welfare Com-
mittee at Benha University’s Faculty of Veterinary
Medicine (BUFVTM 03-02-22).
Birds and experimental design
A total of 150 1-day-old male Cobb broiler chicks were
purchased from El-Huda Company (Al-Dier, Qalyu-
bia, Egypt) for hatching. All chicks were fed a broiler
starter ration from 0-10 days, a grower ration from
11–24 days, and a finisher ration from 25–35 days
(NRC, 1994) (Table 1). Food and water were provided
ad libitum. The chicks were raised on oor pens
bedded by wood shavings in specialized facilities at
the Faculty of Veterinary Medicine, Benha University,
Egypt, following the best management practices guide-
lines. The bedding material was covered by plastic
sheets from 14–24 days of age to facilitate dropping
scoring and collection for oocysts counting. The
chicks were provided with all necessary living con-
ditions throughout the experiment, including heaters
and humidity.
The chicks were weighed and randomly divided
into five groups (six chicks/replicate; five repli-
cates/group) as follows: G1-Ng: fed basal diet with-
out challenge (control negative); G2-Ps: fed basal
diet and challenged with Eimeria spp. oocysts (con-
trol positive); G3-Clo: challenged and fed a basal
diet with clopidol (Atco-pharma, Quisna, Egypt)
at a rate of 0.9 g/kg following the recommen-
dations of the manufacturer; G4-NOa: challenged
and fed a basal diet with N. oculata at a rate of
0.1% (1 g/kg diet); and G5-NOb: challenged and
fed a basal diet with N. oculata at a rate of 0.2%
(2 g/kg diet).
Table 1. Composition of basal diets.
Item
%
Starter
(0–10 d)
Grower
(11–24 d)
Finisher
(25–35 d)
Yellow corn 56.92 60.22 65.44
Soybean meal 46% 35 33.50 28.20
Corn gluten meal 2.3 – –
Vegetable oil 0.95 2 2.30
Sodium chloride 0.22 0.26 0.24
DL-Methionine 0.36 0.34 0.34
L-Lysine 0.40 0.22 0.26
Limestone 1.67 1.55 1.44
Mono-calcium phosphate 1.20 1.025 0.90
Sodium bicarbonate 0.30 0.20 0.23
Vit. and mineral premix 0.30 0.30 0.30
Calculated nutritional value
Crude protein % 23.03 21.02 19.01
MEn (Kcal/kg) 3009.47 3102.61 3198.84
Crude fat % 3.51 4.56 4.99
Lysine % 1.40 1.20 1.10
Lysine digestible % 1.28 1.09 1.00
Methionine % 0.69 0.63 0.60
Methionine digestible % 0.66 0.60 0.58
Methionine + cystine % 1.03 0.95 0.90
Methionine + cystine digestible % 0.94 0.87 0.83
Threonine % 0.95 0.88 0.75
Threonine digestible % 0.83 0.77 0.65
Calcium % 0.96 0.88 0.80
Avail. phosphorus % 0.49 0.44 0.40
Chloride % 0.24 0.23 0.23
Sodium % 0.17 0.16 0.16
Potassium % 0.88 0.85 0.76
2 M. I. ABDEL HALEEM ET AL.
Experimental challenge with coccidiosis
The Eimeria oocysts used in the current study were
collected from commercial broiler houses at Qalyubia
Governorate, Egypt (30° 24′36′′ N, 31° 12′36′′ E). The
oocysts were separated through sieving and sedimen-
tation techniques (Longstae, 1984). The culture was
inspected under a microscope to distinguish the
types of existing oocysts based on morphological
characteristics (Al-Gawad et al., 2012). Five Eimeria
spp. were identified: E. tenella (65%), E. acervulina
(10%), E. maxima (10%), E. necatrix (10%) and
E. mitis (5%). Preliminary laboratory challenge of
broiler chicks validated the culture’s capacity to pro-
duce a characteristic picture of coccidiosis (Swayne
et al., 2020), and the oocysts were found to be viable.
The suspension of fresh sporulated oocysts was stored
in a 2.5% potassium dichromate solution in a refriger-
ator (4°C) until use. On day 14 of the experiment, the
number of sporulated oocysts in the culture was
counted and identified microscopically. Fresh sporu-
lated oocysts (5 × 10
3
oocysts/chick) were adminis-
tered intracrop to groups 2, 3, 4, and 5 with a
volume of 1.5 ml (Pop et al. 2019). The negative con-
trol group received 1.5 ml of normal saline solution as
an inoculant.
Nannochloropsis oculata preparation and
evaluation
N. oculata powder was purchased from the National
Research Center, Dokki, Giza, Egypt. The total pheno-
lic content of N. oculata powder was determined
(Makkar et al., 1997). Its total avonoid content was
estimated (Ordoñez et al., 2006). Standardization in
methanol with quercetin (1 mg/ml) yielded a linear
relationship (y = 0.0071x; R
2
= 0.9979). Quercetin
equivalents (mg QE/g of dry mass) were used to quan-
tify the total avonoid content because it is a widely
used reference ingredient. Each experiment was per-
formed three times to ensure accuracy.
The carotenoid content of N. oculata was estimated
spectrophotometrically (Lichtenthaler & Buschmann,
2001), and its condensed tannins were also deter-
mined (Glick & Joslyn, 1970).
Clinical parameters
Bird droppings were scored from 0-4 (Morehouse
and Barron, 1970) from 0-10 days post-infection
(dpi) based on the consistency of the faeces and the
presence of mucus and/or blood as follows: a score
of 0 indicated normal colour and consistency; a
score of 1 indicated mild mucoid to watery drop-
pings; a score of 2 indicated moderate mucoid to
watery droppings with abnormal colour; a score of
3 indicated all watery, bloody tinged droppings; and
a score of 4 indicated watery bloody droppings.
Macroscopic lesion scores of the duodenum, jeju-
num, and cecum were also observed and recorded
at 10 dpi (Ali et al., 2014).
Oocyst shedding was estimated in fresh faecal
samples (five samples per group) collected from 3
and 10 dpi. The faecal samples were stored separately
in airtight plastic bags, homogenized, and kept at 4°C.
The oocyst count was calculated using a McMaster
counting chamber and represented as oocysts per
gram faeces (OPG) (Bortoluzzi et al., 2018).
Data on feed intake (FI), body weight (BW), and
weight gain (WG) were recorded from day zero to ten
dpi. The feed conversion ratio (FCR) per chick was
determined (Abdel Haleem, Hassan et al., 2019) as fol-
lows:$^id#>
FCR =Average FI (g) per chick in period
Average WG(g) perchick in thesame period
The following formula (Abdel Haleem, Hassan et al.,
2019) was used to obtain the mortality rate:$^id#>
Mortality rate (%) =
Number of deaths in a specified period
Total population during that period ×100
Meat quality characteristics
Fatty acid profile
Five breast muscles (Musculus pectoralis superficialis)
from each group were dissected. The fatty acid
profile of meat was determined using a colourimetric
method according to a previously described technique
(El-bahr et al., 2021) with some modifications. In
brief, samples of chicken breast meat were homogen-
ized and centrifuged to produce a tissue homogenate
(1792 × g for 15 min). The obtained supernatant was
then used to determine the total lipids and total
cholesterol using a commercial kit (Stanbio Labora-
tory Company; Boerne, TX, USA). The total lipid con-
tent was extracted from breast esh samples by
centrifuging them at 1792 × g for 10 min after vortex-
ing for 2 min in chloroform: methanol (2:1; v/v) sol-
ution. Fatty acid methyl esters (FAMEs) were
produced from the supernatant using a methanol: sul-
phuric acid mixture (95:5; v/v) and hexane after the
esterification reaction. Gas chromatography (GC; Agi-
lent Technologies, Santa Clara, CA, USA) with an
SP2330 column (30 mm × 0.32 mm × 0.2 µm film
thickness; Supelco Analytical, St. Louis, MO, USA)
and ame ionization detector was used to analyze
the FAME hexane extract using a temperature gradi-
ent programme, hydrogen as the carrier gas, and a
split model. The retention time of the fatty acid stan-
dard (Cat. No. 24073, Sigma‒Aldrich, St. Louis, MO,
USA) was compared to the retention time of the
AVIAN PATHOLOGY 3
FAME peaks in Hewlett-Packard ChemStation soft-
ware (Agilent Technologies Inc.).
Breast meat physicochemical properties
The pH of the chicken breast muscles from each treat-
ment was measured in triplicate using a pH meter
(Jenway 3510 pH meter, Cole-Parmer, St. Neots,
UK) 24 h post-mortem (ultimate pH (pHu)). The
measurements were made with a portable pH metre
equipped with a glass electrode. To determine the
water holding capacity (WHC), the low-speed cen-
trifugation method was used. Briey, 5 g of intact
breast muscle was trimmed and weighed and then
placed in a Falcon tube with glass beads at 10,000 × g
at 5°C for 20 min. After centrifugation, the trimmed
breast meat was removed immediately, dried with
filter paper and weighed again. The percentage of
muscle weight loss after centrifugation was calculated
for the WHC (Honikel & Hamm, 1994). The drip and
cooking losses (DL&CL) were evaluated (Honikel,
1998). In a closed plastic container, a small cube of
chicken breast muscle (60 ± 2 g) was placed over a
grid. After 24 and 48 h of storage at 4°C, the drip
loss (DL
24
and DL
48
) was calculated as a percentage
of weight loss during this period. The thawing loss
of chicken breast samples was measured (Sun et al.,
2019). The frozen meat samples were thawed in the
refrigerator at 4°C and then dried using filter paper
to remove the surface water. The thawing loss was cal-
culated from the weights of the breast meat samples
before (M0) and after (MT) thawing (Xia et al.,
2009):$^id#>
Thawing loss (%) =(M0 −MT)/M0 ×100
For cooking loss determination (Xia et al., 2012), indi-
vidually weighed chicken breast samples from each
treatment were wrapped in plastic bags and cooked
in an 80°C water bath for 1 h until the internal temp-
erature reached 75°C. Then, the samples were sud-
denly cooled in crushed ice to 5°C and carefully
blotted with filter paper before reweighing. Cooking
loss was determined by weighing the samples before
(M0) and after (Mc) cooking:$^id#>
Cooking loss (%) =(M0 −MC)/M0 ×100
The Warner‒Bratzler shear force (WBSF) was then
calculated using 3343 Universal Test System Mono
columns (Instron, Pune, India) (Silva et al., 2017).
High performance liquid chromatography (HPLC)
(Agilent HP 1200 series apparatus) was used to
measure malondialdehyde (MDA) following protocols
outlined earlier (El-Bahr et al., 2020).Using a Chroma
Meter CR-410 (Konica Minolta Sensing INC., Osaka,
Japan), the colour of the meat was measured using
the CIE lightness (L*), redness (a*), and yellowness
(b*) systems 24 h after slaughter. Meat samples were
scanned at five separate sites for colour measurement.
Clinicopathological parameters
Blood samples (five/group) were collected at 14, 21
and 28 days of age from the right jugular vein of the
neck using sterile needles to separate serum, which
was used for further analyses.
Oxidative stress biomarkers were determined, such as
antioxidant enzymes in serum (Biodiagnostic kit’s guide-
lines), serum MDA using a colorimetric technique (Kei,
1978), serum total superoxide dismutase (T-SOD)
(Nishikimi et al., 1972) and catalase enzyme (Aebi, 1984).
Serum biochemical parameters were evaluated, such
as serum aspartate transaminase (AST) and alanine
transaminase (ALT) (Young, 1997), high-density lipo-
protein cholesterol (HDL-C) (Lopes-Virella et al.,
1977), low-density lipoprotein cholesterol (LDL-C)
(Wieland & Seidel, 1983), total cholesterol (Richmond,
1973), triacylglycerol (Fossati & Prencipe, 1982), and
creatinine and urea (Tovar et al., 2002).
Transmission electron microscopy (TEM)
Five caeca per group were collected and preserved in
glutaraldehyde at 10 dpi for electron microscope
examination. The stained grids were examined and
photographed using a JEOL, JEM-1400-EXELEC-
TRON MICROSCOPE at Ain Shams University’s
Central Laboratory using uranyl acetate and lead
citrate (Ball et al., 2014).
Molecular docking
Molecular docking of clopidol, as a reference drug, and
N. oculata bioactive compounds with E. tenella aldo-
lase, E. tenella apical membrane antigen 1 (EtAMA1),
and E. tenella microneme protein 3 (EtMIC3) were
assessed after retrieval of the three-dimensional struc-
tures of ALD, EtAMA1, and EtMIC3 from AlphaFold
(https://alphafold.ebi.ac.uk/) protein structure data-
bases, while clopidol and N. oculata bioactive com-
pounds were retrieved from LOTUS: Natural
Products Online (https://lotus.naturalproducts.net/)
and PubChem (https://pubchem.ncbi.nlm.nih.gov/)
databases. Three-dimensional structures of E. tenella
aldolase, EtAMA1, and EtMIC3 were prepared and
subjected to molecular docking with N. oculata bio-
active compounds using Molecular Operating
Environment (MOE 2015.10, Chemical Computing
Group, Montreal, QC, Canada) software with the
induced fit method. Additionally, MOE was used to
determine and visualize the protein‒ligand inter-
actions (Vilar et al., 2008).
4 M. I. ABDEL HALEEM ET AL.
Statistical analysis
Analysis of variance (general linear model) was per-
formed on experimental data using the statistical
application SPSS version 20.0 for Windows (SPSS,
Inc., Chicago, IL, USA). Tukey’s post-hoc test (P <
0.05) was conducted to determine the significance
level between the experimental groups.
Results
Phytochemical analyses
The extract of N. oculata contained many active com-
pounds, such as total phenols (39.59 mg gallic acid/g),
avonoids (8.41 mg quinstine/g), carotenoids (3.85 mg/
g), and condensed tannins (0.45 mg tannic/g) (Table 2).
Clinical evaluation
The positive control (G2-Ps) recorded a remarkable
(P < 0.05) increase in scores of droppings (0.33–3.00)
when compared to the G1-Ng (0.00). The highest
score of droppings was recorded in G2-Ps (2.33 and
3.00, respectively) in comparison to the other chal-
lenged groups. A profound (P < 0.05) decline in drop-
ping score was recorded in G3-Clo at 5 dpi (0.00) and
in groups G3-Clo, G4-NOa, and G5-NOb at 6 dpi
(1.00, 0.33, and 1.00, respectively) when compared to
G2-Ps. G4-NOa recorded a numerical (P > 0.05)
decline (0.33) in scoring at 6 dpi (Table 3 and Figure
2).
Microscopic examination of the droppings revealed
the presence of coccidial oocysts from 4-7 dpi (Figure
1). No oocysts were recorded in the droppings of G1-
Ng. The OPG in G2-Ps dramatically increased from
that of G1-Ng (P < 0.05). A nonsignificantly (P >
0.05) number of oocysts was observed in the treated
groups compared to G1-Ng. The OPG in the G5-
NOb and G3-Clo groups was significantly lower
than that of the G2-Ps at 5, 6, and 7 dpi (P < 0.05;
4351, 4313.3, and 689.00, respectively). On day 7,
G4-NOa had significantly fewer oocysts (31.00) than
G2-Ps (689.00) (P < 0.05).
The macroscopic findings of the duodenum, jeju-
num, and caecum were notably (P < 0.05) increased
(1.20, 1.00, and 2.40, respectively) in G2-Ps compared
to G1-Ng (Table 4 and Figure 2). There was a remark-
able (P < 0.05) decline in the scores of the duodenum,
jejunum, and caecum in treated challenged birds (G4-
NOa, G5-NOb, and G3-Clo) in comparison to those of
G2-Ps. The scores of the duodenum and caecum in
groups G4-NOa and G5-NOb were numerically (P >
0.05) lower than those of G3-Clo. The jejunum of
G4-NOa and G5-NOb showed a remarkable (P <
0.05) decline in the lesion when compared to G3-Clo
(1.00).
The FI at 10 dpi was not profoundly (P > 0.05)
dierent in the challenged groups, whether treated
or not, in comparison with the G1-Ng group (840 g);
likewise, the situation for BW and WG values during
that period of the study. Regarding the FCR, G1-Ng
recorded the lowest value (1.72), but this result was
not profoundly (P > 0.05) dierent from the chal-
lenged and/or treated groups. No mortalities were
recorded in the dierent groups of the experiment
during the relevant study period (Table 5).
Meat quality characteristics
The change in fatty acid composition (g/100 g muscle)
caused by dietary supplementation with N. oculata in
the breast muscles of coccidiosis-challenged broilers
is shown in Table 6. The concentrations of eicosapen-
taenoic acid (C20:5n-3, EPA), α-linolenic acid (ALA,
C18:3n-3), and total ω −3 polyunsaturated fatty acid
(PUFA) in G5-NOb were markedly (P < 0.05) elevated
in comparison to other groups. However, N. oculata
microalgae inuence EPA levels but do not inuence
docosahexaenoic acid (C22:6n-3, DHA) levels. The
increase in EPA content subsequently increased the
Table 2. Chemical characterization of dried Nannochloropsis
oculata powder.
Compounds Total
phenols
(mg gallic/
g D.W.)
Total
flavonoids
mg
Quercetin/
gD.W.
Total
carotene
(mg/g)
Condensed
tannins
(mg tannic/
g D.W.)
39.59±0.17 8.41±0.17 3.85±0.09 0.45±0.04
Values are given as the mean (n = 5) ± standard deviation (absolute
value).
Table 3. Effect of Nannochloropsis oculata and clopidol on dropping scoring of Eimeria spp. challenged broiler chicks.
Group 3 dpi 4 dpi 5 dpi 6 dpi 7 dpi 8 dpi 9 dpi 10 dpi
G1-Ng 0.00
c
0.00
b
0.00
b
0.00
b
0.00
a
0.00
b
0.00
b
0.00
a
G2-Ps 1.67
a
1.67
a
2.33
a
3.00
a
1.00
a
1.00
a
0.67
a
0.33
a
G3-Clo 1.67
a
0.67
ab
0.00
b
1.00
b
0.67
a
0.33
b
0.00
b
0.00
a
G4-NOa 0.33
bc
0.67
ab
0.67
ab
0.33
b
1.00
a
0.00
b
0.00
b
0.00
a
G5-NOb 1.00
ab
0.67
ab
1.00
ab
1.00
b
0.00
a
0.00
b
0.00
b
0.00
a
SEM 0.149 0.067 0.221 0.133 0.115 0.115 0.094 0.067
P value 0.015 0.00 0.035 0.001 0.147 0.024 0.072 0.452
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–c
Means within a column not sharing a common
superscript differ significantly when P < 0.05. SEM: standard error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal diet without challenge;
G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata; G5-
NOb: challenged and fed 0.2% N. oculata. dpi: day post-infection.
AVIAN PATHOLOGY 5
total PUFA/SFA ratio compared to that in the G1-Ng
group. In addition, total EPA+DHA was not notably
(P > 0.05) altered among the experimental groups, but
higher concentrations were recorded in the
microalgae-supplemented groups (0.1 and 0.2%) than
in the other groups. Regarding meat quality attributes,
WHC, DL (24 and 48 h), thawing loss, and cooking loss
parameters of broiler breast meat samples dramatically
(P < 0.05) diered among treatments (Table 7). Treat-
ment with 0.2% N. oculata profoundly (P < 0.05)
aected the WHC. As the microalgae level treatment
increased, the WHC increased, leading to improved
meat quality. However, a notable (P < 0.05) decrease
in drip loss (after 24 and 48 h) of breast meat was
observed in the G5-NOb group compared to the
other groups. The thawing loss and cooking loss
showed substantially (P < 0.05) the highest values in
G3-Clo compared to the other groups (Table 7). How-
ever, other meat quality parameters, such as WBSF and
colour characteristics (L*, a*, and b*), remained
remarkably (P > 0.05) altered in all experimental
groups. Furthermore, the G4-NOa and G5-NOb
groups showed a marked (P < 0.05) reduction in
MDA in breast meat compared to the G2-Ps group,
and nonprofound changes (P > 0.05) compared to the
G1-Ng and G3-Clo groups (Table 7).
Clinicopathological parameters
Oxidative stress parameters
The MDA values increased gradually in the challenged
groups through the period of the experiment (14, 21,
and 28 days) and reached 4.28 ± 0.09 nmol/ml after
28 days in G5-NOb, while the control group recorded
2.45 nmol/ml. Both treatments of N. oculata extract
(0.1 and 0.2%) increased the MDA values, as in the
case of G3-Clo. The highest values of MDA were
recorded in G5-NOb (2.75 nmol/ml) and G3-Clo
(2.70 nmol/ml). The T-SOD value profoundly (P <
0.05) decreased in G2-Ps in comparison with the chal-
lenged treated groups. The highest alleviation rate was
found in the G3-Clo and G5-NOb groups, followed by
the G4-NOa group. Using clopidol and 0.2%
N. oculata extract in diets resulted in values similar
to those of G1-Ng at the end of the experiment
(119.42 U/mg), which confirmed the eciency of the
Figure 1. Effect of Nannochloropsis oculata and clopidol on oocyst shedding in droppings (OPG) of Eimeria spp. challenged broiler
chicks.
Table 4. Effect of Nannochloropsis oculata and clopidol on
intestinal lesion scoring of Eimeria spp. challenged broiler
chicks.
Group/Organ Duodenum Jejunum Caecum
G1-Ng 0.00
b
0.00
c
0.00
c
G2-Ps 1.20
a
1.00
a
2.40
a
G3-Clo 0.40
b
0.40
b
1.40
b
G4-NOa 0.20
b
0.00
c
0.40
bc
G5-NOb 0.00
b
0.00
c
0.40
bc
SEM 0.075 0.049 0.147
P-value 0.00 0.00 0.00
Tukey’s test represents the least profound differences between different
groups at probability P < 0.05.
a–c
Means within a column not sharing
a common superscript differ significantly when P < 0.05. SEM: standard
error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal
diet without challenge; G2-Ps: challenged with Eimeria spp. and fed
basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: chal-
lenged and fed 0.1% N. oculata; G5-NOb: challenged and fed 0.2%
N. oculata.
6 M. I. ABDEL HALEEM ET AL.
treatments. Concerning catalase results, a slight
increase was recorded in the G2-Ps, while no profound
(P > 0.05) eect was noticed in the other treated chal-
lenged groups (Table 8).
Lipid profile
Both total cholesterol and triacylglycerols were dra-
matically (P < 0.05) decreased after challenge with
Eimeria spp. when compared to G1-Ng at 28 days of
age (Table 9). A considerable increase was noted in
the levels of total cholesterol and triacylglycerols in
both N. oculata-treated groups as well as the G3-Clo
group. The highest increase in total cholesterol and
triacylglycerols was recorded at 28 days in both G5-
NOb and G3-Clo, followed by G4-NOa. Similarly,
HDL and LDL cholesterol notably (P < 0.05) decreased
after challenge with Eimeria spp. G5-NOb, G4-NOa,
and G3-Clo exhibited a promising increase in HDL
values at 28 days (65.78, 60.45, and 61.11 mg/dl,
respectively) even more than G1-Ng. LDL cholesterol
slightly increased as a response to infection with
Eimeria spp., but no profound (P > 0.05) variation
was noticed between dierent treatments.
Liver and kidney functions
AST and ALT values in the G2-Ps group were pro-
foundly (P < 0.05) increased (almost twofold) after
the challenge in comparison to G1-Ng (Table 10).
The highest improvement was recorded in G3-Clo,
followed by G5-NOb. Regarding kidney function mar-
kers, the results revealed that serum urea of the chal-
lenged broilers dramatically (P < 0.05) increased in
G2-Ps in comparison to the G1-Ng group. However,
it was markedly (P < 0.05) lower in the G3-Clo
group than in the G2-Ps group. Additionally, the
serum urea content was reduced by 5.17 mg/dl in
G5-NOb. No (P < 0.05) variations were noted in
serum creatinine between dierent groups during
the experimental period.
Transmission electron microscopy
The negative control (G1-Ng) intestinal cells had nor-
mal architecture, with well-developed tight junctions
connecting them (Figure 3A). Moreover, the entero-
cyte supranuclear region contained several
Table 5. Effect of Nannochloropsis oculata and clopidol on the
performance of Eimeria spp. challenged broiler chicks.
Group/ Parameter FI (g) BW (g) WG (g) FCR Mortality (%)
G1-Ng 840
a
733.33
a
494.67
a
1.72
a
0
G2-Ps 806.67
a
723.33
a
477.67
a
1.73
a
0
G3-Clo 843.33
a
723.33
a
487
a
1.74
a
0
G4-NOa 910
a
713.33
a
469.33
a
1.94
a
0
G5-NOb 846.67
a
740.00
a
489.67
a
1.74
a
0
SEM 16.289 10.667 9.133 0.045 –
P value 0.425 0.941 0.906 0.445 –
Tukey’s test represents the least profound differences between different
groups at probability P < 0.05.
a-c
Means within a column not sharing
a common superscript differ significantly when P < 0.05. SEM: standard
error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal
diet without challenge; G2-Ps: challenged with Eimeria spp. and fed
basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: chal-
lenged and fed 0.1% N. oculata; G5-NOb: challenged and fed 0.2%
N. oculata. FI: feed intake; BW: body weight; WG: weight gain; FCR:
feed conversion ratio.
Figure 2. Pathological changes in droppings and the intestinal tract of Eimeria spp. challenged birds. a: normal colour and consistency
droppings (score 0); b: watery dark red to brown droppings (score 4); c: Numerous petechiae on the serosal surface of the duodenum
(right) and jejunum (left); d: mucoid inflammation with petechial haemorrhage on the small intestine mucosal surface; e: numerous red
and white foci on the caecum’s serosal surface; f: normal caecum mucosa (right), inflamed oedematous mucosa with petechial haem-
orrhages (left); g: severe haemorrhagic inflammation, mucosal debris, and blood in the caecal lumen, with a thickened wall.
AVIAN PATHOLOGY 7
mitochondria and numerous rough endoplasmic cis-
ternae. Furthermore, the luminal surface of the
enterocytes was densely packed with parallel finger-
like microvilli. The intestinal cells of G2-Ps showed
infection in the caecal epithelium with scant
cytoplasm. Moreover, several swollen (or damaged)
cytoplasmic organelles and various cytoplasmic
inclusions of Eimeria oocytes in either mature or
first-generation schizonts in a more advanced stage
of merozoite formation were recorded (Figure 3B
Table 6. Effect of Nannochloropsis oculata and clopidol on the fatty acid profile of breast meat in Eimeria spp. challenged broiler
chicks.
Fatty acids (g/100 g) tissue/Groups G1-Ng G2-Ps G3-Clo G4-NOa G5-NOb SEM P-value
Myristic
C14:0
0.75
bc
0.90
a
0.92
a
0.71
b
0.73
b
0.016 0.004
Palmitic
C16:0
27.70
a
24.23
c
23.63
c
25.47
bc
26.76
ab
0.297 0.007
Stearic
C18:0
11.80 11.56 12.43 11.26 10.89 0.226 0.325
Myristovaccenic
C14:1
0.14 0.14 0.15 0.13 0.14 0.003 0.419
Palmitoleic
C16:1
4.26
a
3.69
b
3.57
b
3.98
ab
3.84
ab
0.064 0.051
Oleic
C18:1
30.22 33.25 32.72 30.79 30.20 0.515 0.255
Linoleic (LA)
C18:2 (n−6)
14.95
b
16.52
a
16.64 17.61 16.60 0.221 0.048
Gamma-linolenic (GLA)
C18:3 (n-6)
1.93 1.73 1.97 1.73 1.92 0.106 0.913
Eicosadienoic (EDA)
C20:2 (n-6)
0.97 0.98 0.92 1.03 1.02 0.028 0.73
Arachidonic (AA)
C20:4 (n-6)
3.60
a
2.96
b
3.00
b
3.49
a
3.56
a
0.037 000
Docosadienoic
C22:2 (n-6)
1.37 1.42 1.33 1.57 1.72 0.061 0.326
Adrenic
C22:4 (n-6)
1.47 1.74 1.59 1.54 1.68 0.078 0.824
α-Linolenic acid (ALA)
C18:3 (n-3)
0.31
b
0.35
ab
0.33
b
0.34
ab
0.38
a
0.006 0.032
Docosahexaenoic (DHA)
C22:6 (n-3)
0.26 0.29 0.27 0.31 0.29 0.01 0.569
Eicosapentaenoic (EPA)
C20:5 (n-3)
0.26
ab
0.22
c
0.23
bc
0.26
abc
0.28
a
0.005 0.031
∑ SFA 40.26
a
36.69
b
36.98
b
37.43
b
38.39
ab
0.376 0.035
∑ MUFA 34.62 37.09 36.43 34.90 34.18 0.519 0.384
∑ PUFA 25.13
b
26.22
ab
26.59
ab
27.67
a
27.44
ab
0.33 0.041
∑ n-3 PUFA 0.84
ab
0.87
ab
0.83
b
0.90
ab
0.95
a
0.015 0.027
∑ n-6 PUFA 24.29
b
25.35
ab
25.76
ab
26.76
a
26.48
ab
0.316 0.032
PUFA/SFA ratio 0.62
b
0.71
a
0.72
a
0.74
a
0.72
a
0.011 0.047
∑ EPA+DHA 0.52 0.51 0.51 0.57 0.57 0.013 0.401
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–c
Means within a row not sharing a common
superscript differ significantly when P < 0.05. SEM: standard error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal diet without challenge;
G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata; G5-
NOb: challenged and fed 0.2% N. oculata. ∑SAF: total saturated fatty acid; ∑PUFA: total polyunsaturated fatty acid; ∑ MUFA: total monounsaturated
fatty acid; Σn−3 PUFA: total omega 3 polyunsaturated fatty acid; and Σn−6 PUFA: total omega 6 polyunsaturated fatty acid.
Table 7. Effect of Nannochloropsis oculata and clopidol on breast meat physicochemical properties in Eimeria spp. challenged
broiler chicks.
Parameters groups G1-Ng G2-Ps G3-Clo G4-NOa G5-NOb SEM P value
Ultimate pH (pH24) 5.38 5.45 5.46 5.49 5.50 0.024 0.538
WHC 89.61
a
82.24
b
81.97
b
82.94
b
88.75
a
0.927 0.043
DL24 11.70
a
12.30
a
10.67
ab
9.62
ab
7.67
b
0.884 0.041
DL48 13.20
a
12.89
a
12.07
ab
11.42
ab
9.09
b
0.857 0.039
Thawing loss 2.21
b
2.94
b
14.49
a
1.43
b
0.87
b
0.249 0
Cooking loss 7.79
b
9.67
ab
11.75
a
8.59
b
8.56
b
0.610 0.036
WBSF 3.26 3.59 3.34 3.58 3.38 0.072 0.631
MDA 20.62
b
28.02
a
22.38
ab
20.31
b
19.10
b
0.912 0.007
CLAB coordinate
L* 51.78 50.85 51.68 51.33 53.63 1.091 0.510
a* 11.54 11.26 11.32 11.44 11.01 0.264 0.825
b* 8.37 10.33 10.09 8.65 9.97 0.486 0.563
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–c
Means within a row not sharing a common
superscript differ significantly when P < 0.05. SEM: standard error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal diet without challenge;
G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata; G5-
NOb: challenged and fed 0.2% N. oculata. WHC: water holding capacity; DL
24
: drip loss after 24 h; DL
48
: drip loss after 48 h; WBSF: Warner-Bratzler Shear
Force; L*: lightness; a*: redness; b*: yellowness; MDA: malondialdehyde.
8 M. I. ABDEL HALEEM ET AL.
and C). G3-Clo intestinal cells exhibited destruction
of enterocyte brush borders as well as the presence of
various Eimeria spp. developmental stages. The
number of protozoan development stages was
lower in G3-Clo than in G2-Ps (Figure 3D and E).
The intestinal cells of 0.1 and 0.2% N. oculata
treatments displayed round or elongated mitochon-
dria with little or no structural damage. In G5-
NOb, the number of developmental stages of Eimeria
spp. in the absorptive epithelial lining of intestinal
villi was dramatically lower than that in G3-Clo
and G4-NOa (Figure 3F and G). Furthermore,
degenerated parasitic stages were observed in G5-
NOb enterocytes (Figure 3H and I).
Molecular docking
Clopidol exhibited binding energies to E. tenella aldolase
(Figure 4a), EtAMA1 (Figure 5a), and EtMIC3 (Figure
6a) of −6.45, −4.56, and −4.60 kcal/mol, respectively.
N. oculata bioactive compounds, violaxanthin, zeax-
anthin, canthaxanthin, echinenone, (3s)-6-[(1e,3e,5e,
7e,9e,11e,13e,15e,17e)-18-[(4r)-4-hydroxy-2,6,6-tri-
methylcyclohex-1-en-1-yl]-3,7,12,16-tetramethylo-
ctadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl]-1,5,5-tri-
methyl-7-oxabicyclo[4.1.0]heptan −3-ol and luteox-
anthin interacted with the E. tenella aldolase binding
site with binding energies of −6.45 (Figure 4b),
−6.88 (Figure 4c), −7.23 (Figure 4d), −6.73 (Figure
4e), −6.75 (Figure 4f), and −6.71 kcal/mol (Figure
Table 8. Effect of Nannochloropsis oculata and clopidol on oxidative stress biomarkers in the serum of Eimeria spp. challenged
broiler chicks.
Group/Parameter
MDA (nmol/ml) T-SOD (U/mg) Catalase (U/mg)
days days days
14 21 28 14 21 28 14 21 28
G1-Ng 2.08
d
2.45
c
2.44
b
118.17
a
121.42
a
119.42
a
13.14
b
13.15
b
12.69
b
G2-Ps 2.93
a
3.67
a
4.28
a
100.44
e
98.76
e
82.93
d
14.04
a
14.09±0.08
a
13.70
a
G3-Clo 2.24
c
2.58
c
2.70
b
111.88
b
115.07
b
118.00
ab
13.26
ab
12.84±0.10
bc
12.65
b
G4-NOa 2.54
ab
3.11
b
3.94
a
103.20
d
102.44
d
97.43
c
12.98
b
12.56
c
12.51
b
G5-NOb 2.44
ab
2.77
bc
2.75
b
108.03
c
113.65
c
117.57
b
13.13
b
13.23
b
12.59
b
SEM 0.019 0.050 0.050 0.217 0.183 0.204 0.118 0.074 0.064
P-value 0.00 0.00 0.00 0.00 0.00 0.00 0.110 0.00 0.00
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–c
Means within a column not sharing a common
superscript differ significantly when (P < 0.05). SEM: standard error of mean. Values are given as the mean (n = 5).G1-Ng: fed basal diet without chal-
lenge; G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata;
G5-NOb: challenged and fed 0.2% N. oculata. MDA: malondialdehyde; T-SOD: total superoxide dismutase.
Table 9. Effect of Nannochloropsis oculata and clopidol on the lipid profile in the serum of Eimeria spp. challenged broiler chicks.
Group/Parameter
Total cholesterol (mg/dl) HDL-C (mg/dl) LDL-C (mg/dl) Triacylglycerol (mg/dl)
days days days days
14 21 28 14 21 28 14 21 28 14 21 28
G1-Ng 162.73
a
164.52
a
165.26
a
60.96
a
59.23
b
61.12
b
36.01
a
36.25
a
35.60
a
99.07
a
104.60
a
110.09
ab
G2-Ps 141.23
bc
138.10
d
129.07
d
48.90
d
44.93
d
40.78
c
22.42
d
22.95
c
22.72
d
75.56
b
75.22
c
70.44
c
G3-Clo 141.94
b
145.59
bc
163.29
b
58.11
b
58.65
b
61.11
b
24.77
b
24.27
b
25.30
b
99.45
a
101.26
b
110.58
ab
G4-NOa 140.11
c
144.60
c
161.41
c
54.71
c
54.60
c
60.44
b
22.40
d
22.42
c
22.45
d
97.50
a
100.07
b
109.23
b
G5-NOb 140.78
bc
145.82
b
163.67
b
58.71
b
62.71
a
65.77
a
23.60
c
23.90
b
24.08
c
98.92
a
101.42
b
112.14
a
SEM 0.238 0.158 0.153 0.151 0.178 0.121 0.118 0.081 0.104 0.298 0.272 0.281
P-value 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–c
Means within a column not sharing a common
superscript differ significantly when P < 0.05. SEM: standard error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal diet without challenge;
G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata; G5-
NOb: challenged and fed 0.2% N. oculata. HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol.
Table 10. Effect of Nannochloropsis oculata and clopidol on liver and kidney functions in the serum of Eimeria spp. challenged
broiler chicks.
Group/Parameter
AST (U/l) ALT (U/l) Urea (mg/dl) Creatinine (mg/dl)
days days days days
14 21 28 14 21 28 14 21 28 14 21 28
G1-Ng 55.75
e
56.15
e
56.47
e
128.20
d
129.30
d
128.82
e
4.25
a
4.37
c
4.72
d
0.38
b
0.40
a
0.41
ab
G2-Ps 85.96
a
91.28
a
110.03
a
151.89
a
155.69
a
202.95
a
4.60
a
5.03
a
6.40
a
0.39
b
0.40
a
0.42
ab
G3-Clo 76.87
c
75.97
c
63.26
c
131.35
c
145.66
b
142.81
d
4.39
a
4.92
ab
4.82
cd
0.41
a
0.41
a
0.42
ab
G4-NOa 82.71
b
81.03
b
81.37
b
142.31
b
141.22
c
197.62
b
4.23
a
4.70
abc
5.71
b
0.39
b
0.40
a
0.40
b
G5-NOb 72.67
d
71.12
d
61.11
d
130.69
c
145.86
b
153.43
c
4.63
a
4.51
bc
5.17
c
0.39
b
0.41
a
0.42
a
SEM 0.224 0.198 0.212 0.162 0.240 0.258 0.592 0.064 0.057 0.002 0.003 0.003
P-value 0.00 0.00 0.00 0.00 0.00 0.00 0.16 0.04 0.00 0.02 0.65 0.12
Tukey’s test represents the least profound differences between different groups at probability P < 0.05.
a–e
Means within a column not sharing a common
superscript differ significantly when P < 0.05. SEM: standard error of mean. Values are given as the mean (n = 5). G1-Ng: fed basal diet without challenge;
G2-Ps: challenged with Eimeria spp. and fed basal diet; G3-Clo: challenged and fed clopidol (0.9 g/kg); G4-NOa: challenged and fed 0.1% N. oculata; G5-
NOb: challenged and fed 0.2% N. oculata. ALT: aspartate transaminase; AST: alanine transaminase.
AVIAN PATHOLOGY 9
4g), respectively. Similarly, the same violaxanthin
(−7.33 and −7.28 kcal/mol), zeaxanthin (−8.32
and −7.64 kcal/mol), canthaxanthin (−8.01 and
−7.69 kcal/mol), echinenone (−7.89 and −7.43 kcal/-
mol), (3s)-6-[(1e,3e,5e,7e,9e,11e,13e,15e,17e)-18-[(4r)-
4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl]-3,7,
12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-non-
aen-1-yl]-1,5,5-trimethyl-7-oxa-bicyclo[4.1.0]hepta-
n-3-ol (−7.41 and −6.64 kcal/mol), and luteoxanthin
(−8.66 and −7.67 kcal/mol) bound with EtAMA1
(Figure 5b–g) and EtMIC3 (Figure 6b–g) binding
sites, respectively.
Discussion
A recent trend has emerged to replace the anticocci-
dial drugs added to poultry feed with natural prep-
arations that are favoured by consumers for their
high safety rates and beneficial eects, in response to
addressing their negative eects of the overuse and
misuse on human or bird health. Therefore, this
study evaluated the eect of dietary Nannochloropsis
oculata as a chemotherapeutic alternative for broilers
challenged with coccidiosis.
The biological eectiveness of Nannochloropsis ocu-
lata is attributed to the presence of proteins, lipids,
EPA, PUFAs, as well as antioxidants such as polyphenols,
carotenoids, and vitamins (Zanella & Vianello, 2020). In
the current study, secondary metabolite levels in
N. oculata powder matched those found in the ethanolic
extract of Nanochloroposis gaditana microalgae, with
total phenols, avonoids, and condensed tannins at
41.53 ± 0.42, 19.82 ± 1.66, and 0.35 ± 0.05 mg/g, respect-
ively, as reported by Kherraf et al. (2017). Our findings
came in agreement with Gessner et al., (2017), who
found that the polyphenol content of N. oculata was 28
mg gallic acid/100 g. N. oculata contained a broad set of
antioxidant compounds, as reported previously by Pater-
son et al. (2023).
Experimental birds showed strong reactions to a
mixture of E. tenella, E. acervulina, E. maxima,
E. necatrix, and E. mitis oocysts, as evidenced by
high dropping scores, severe lesions in the intestine,
and a high number of oocysts in the faeces. Similar
observations were previously recorded (Swayne
et al., 2020). The reported pathological findings in
this study are attributed to the host immune system’s
production of free radical oxidative species in
Figure 3. TEM micrograph of the caecum in Eimeria spp. challenged birds. A: normal enterocyte content, nucleus, mitochondria,
microvilli, and apical junction complex (×2000) in G1-Ng; B, C: several mature microgamonts inside epithelial cells and final mer-
ogony, gamont stages (arrow) (1000× & 2500×) in G2-Ps; D, E: the destruction of the brush border of enterocytes as well as a few
Eimeria stages (arrow) (3000× & 1500×) in G3-Clo; F, G: small number of Eimeria stages inside enterocytes (1500× & 1200×) in G4-
NOa; H, I: enterocytes showed intact brush border and mitochondria in addition to degenerated parasitic stages (3000× & 5000×)
in G5-NOb.
10 M. I. ABDEL HALEEM ET AL.
Figure 4. a: Eimeria tenella aldolase and clopidol molecular interaction; b: Eimeria tenella aldolase and violaxanthin mol-
ecular interaction; c: Eimeria tenella aldolase and zeaxanthin molecular interaction; d: Eimeria tenella aldolase and canthax-
anthin molecular interaction; e: Eimeria tenella aldolase and echinenone molecular interaction; f: Eimeria tenella aldolase
and (3s)-6-[(1e,3e,5e,7e,9e,11e,13e,15e,17e)-18-[(4r)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl]-3,7,12,16-tetramethyloc-
tadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl]-1,5,5-trimethyl-7-oxabicyclo[4.1.0]heptan-3-ol molecular interaction; g: Eimeria
tenella aldolase and luteoxanthin molecular interaction.
AVIAN PATHOLOGY 11
Figure 5. a: Eimeria tenella apical membrane antigen 1 (EtAMA1) and clopidol molecular interaction; b: Eimeria tenella apical mem-
brane antigen 1 (EtAMA1) and violaxanthin molecular interaction; c: Eimeria tenella apical membrane antigen 1 (EtAMA1) and
zeaxanthin molecular interaction; d: Eimeria tenella apical membrane antigen 1 (EtAMA1) and canthaxanthin molecular inter-
action; e: Eimeria tenella apical membrane antigen 1 (EtAMA1) and echinenone molecular interaction; f: Eimeria tenella apical
membrane antigen 1 (EtAMA1) and (3s)-6-[(1e,3e,5e,7e,9e,11e,13e,15e,17e)-18-[(4r)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-
yl]-3,7,12,16-tetramethyloctadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl]-1,5,5-trimethyl-7-oxabicyclo[4.1.0]heptan-3-ol molecular
interaction; g: Eimeria tenella apical membrane antigen 1 (EtAMA1) and luteoxanthin molecular interaction.
12 M. I. ABDEL HALEEM ET AL.
Figure 6. a: Eimeria tenella microneme protein 3 (EtMIC3) and clopidol molecular interaction; b: Eimeria tenella microneme
protein 3 (EtMIC3) and violaxanthin molecular interaction; c: Eimeria tenella microneme protein 3 (EtMIC3) and zeaxanthin
molecular interaction; d: Eimeria tenella microneme protein 3 (EtMIC3) and canthaxanthin molecular interaction; e: Eimeria
tenella microneme protein 3 (EtMIC3) and echinenone molecular interaction; f: Eimeria tenella microneme protein 3 (EtMIC3)
and (3s)-6-[(1e,3e,5e,7e,9e,11e,13e,15e,17e)-18-[(4r)-4-hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl]-3,7,12,16-tetramethyloc-
tadeca-1,3,5,7,9,11,13,15,17-nonaen-1-yl]-1,5,5-trimethyl-7-oxabicyclo[4.1.0]heptan-3-ol molecular interaction; g: Eimeria
tenella microneme protein 3 (EtMIC3) and luteoxanthin molecular interaction.
AVIAN PATHOLOGY 13
response to the invasion of Eimeria species, which
causes pathogenic oxidative stress and an altered eco-
logical oxidative balance (Georgieva et al., 2006).
This study indicated that N. oculata (0.1%)
improved the droppings of challenged chicks
compared to G2-PS. This improvement was compar-
able to that in the clopidol-treated group. Further-
more, the N. oculata (0.1 and 0.2%) resulted in a
clear and substantial improvement in the intestinal
condition, and this improvement was superior to
that of the clopidol-treated group. Compared to the
challenged untreated group, the dietary microalgae
profoundly reduced the number of shed oocysts, simi-
lar to that of the clopidol-treated group. These results
are consistent with an earlier study on Nile tilapia fish
(Oreochromis niloticus) (Abdelghany et al., 2020). The
severity of E. tenella infections and intestinal lipid per-
oxidation can be mitigated by antioxidant chemicals
(Allen et al., 1998). As a result, N. oculata minimized
the harmful eects of oxidative stress and brought
back the natural balance of oxidation in the birds’
intestines after the challenge. This attribution has
been previously confirmed in other research (Mam-
douh et al., 2021; Abd El-Hamid et al., 2022).
Regarding performance parameters, the current
study revealed that at 10 days post-infection, the infec-
tion had no apparent impact when compared to G1-
Ng; thus, we did not observe any significant eects
of interventions on FI, BW, WG and FCR. The present
results are supported by several studies (Long et al.,
2018; Park et al., 2018), indicating that a period of
10 days following infection may not be adequate to
clearly observe the impact of the challenge and treat-
ments on performance.
n3-PUFA are inadequately synthesized by the
human body and must therefore be obtained from
the diet (Cartoni Mancinelli et al., 2022). In this
study, the dietary supplementation of coccidiosis-
challenged broilers with N. oculata (2.0%) could pro-
vide a valuable source of such necessary fatty acids
for humans. A similar study recorded that using
microalgae (0.1%) led to a significant increase in
EPA, DHA, total PUFAs, and arachidonic acid in
the breast muscle of broiler chickens (El-Bahr
et al., 2020). The higher concentration of EPA in
the broiler groups supplemented with microalgae in
this study could be due to the fact that Nannochlor-
opsis spp. is abundant in omega-3 fatty acids, par-
ticularly EPA (Kagan & Matulka, 2015).
Furthermore, the amount of EPA+DHA ingested in
100 g of broiler meat fed marine algae met the rec-
ommended daily intake mentioned in previous
reports (Kang et al., 2005; Molendi-Coste et al.,
2011; Aranceta & Pérez-Rodrigo, 2012). In this
study, the total PUFA/SFA ratios observably
increased in the microalgae-supplemented groups
compared to the control negative group, indicating
improved fatty acid balance in the investigated
tissues.
The present study revealed that the addition of
N. oculata to the diet resulted in enhanced meat qual-
ity indicators, including water holding capacity, drip
loss at 24 and 48 h, cooking loss, and thawing loss,
in the challenged broiler chicks. In addition, there
were no significant dierences observed among the
dietary interventions in the ultimate pH level, which
is consistent with findings from other studies (Rajput
et al., 2014; Liu et al., 2020). The WHC assesses the
ability of meat to keep its water content, either fully
or partially (Hu-Lonergan & Lonergan, 2005). In
the present investigation, the WHC increased notably
in the microalgae treated challenged birds in compari-
son to the G2- pos. These findings align with previous
studies (Qaid et al., 2021; Rajput et al., 2014) and can
be attributed to the microalgae used, which supply the
body with omega-3 PUFA. This nutrient aids in the
formation of a exible lipid bilayer membrane in
muscle cells, resulting in improved tenderness, juici-
ness, firmness, and appearance (Mir et al., 2017;
Kalbe et al., 2019).
The current study observed a significant decrease in
drip loss (at 24 and 48 h) in the G5-NOb group com-
pared to the other treatment groups. Comparable
findings were reported in broiler meat (Long et al.,
2018) and pig meat (Kalbe et al., 2019). The reduction
in drip loss of breast meat which was supplemented
with 0.2% N. oculata can be explained by the concur-
rent increase in WHC. The use of microalgae treat-
ment in challenged birds resulted in a decrease in
both cooking loss and thawing loss compared to G3-
Clo. This finding aligns with previous studies by
Khan et al. (2021) and Šefcová et al. (2021).
The level of tenderness in meat is a crucial factor in
assessing its overall quality. An insignificant (P > 0.05)
change in Warner‒Bratzler Shear Force was observed
in all experimental groups during the current study.
Conversely, an increased dosage of Eimeria oocytes
significantly decreased the tenderness of meat (Cho-
dová et al., 2018). The absence of dierences in muscle
shear force among the experimental groups could be
attributed to the relatively little variation in final pH
observed between the groups.
The colour of meat is often used as an indicator of its
freshness and quality (Uhlířová et al., 2018). The
inclusion of N. oculata in the feed of broilers subjected
to coccidiosis challenge did not have a significant
impact on the colour metrics (P > 0.05), aligning with
the results reported by Chodová et al. (2018). Based
on the MDA analysis, G2-Ps exhibited a significant
(P < 0.05) increase compared to both the negative con-
trol and the groups treated with microalgae. There was
no significant dierence (P > 0.05) in the MDA value
between the birds treated with N. oculata and the nega-
tive control. Prior studies have demonstrated an
14 M. I. ABDEL HALEEM ET AL.
elevation in MDA levels in birds when exposed to coc-
cidiosis, which is attributed to oxidative stress (Geor-
gieva et al., 2006; El-maksoud, 2014).
This study revealed that administering of 0.1 or 0.2%
N. oculata resulted in a notable decrease in the oxi-
dative stress parameters to approach the normal ranges.
The dietary N. oculata (0.2%) profoundly (P < 0.05)
increased the total antioxidant capacity compared to
the control group. The current results were in agree-
ment with a previous study of Abd El-Hamid et al.
(2022). The current results could be attributed to the
fact that microalgae have many natural active ingredi-
ents that can help reduce oxidative stress by boosting
antioxidant enzymes and other molecules (Abdelnour
et al., 2020; Abd El-Hamid et al., 2022).
The present investigation found that the N. oculata
treatment eectively minimized the adverse eects on
the lipid profile of the challenged birds. Moreover,
N. oculata (0.1 or 0.2%), improved the quality of the
cholesterol by increasing the DHL. These outcomes
may be explained by the fact that N. oculata contains
polysaccharides that inhibit the intestinal absorption
of total cholesterol and triacylglycerols (Niewold
et al., 2012). Using N. oculata in feed of challenged
birds has improved HDL-C and decreased LDL-C,
possibly due to its anti-hypercholesterolaemia impacts
(Andrés et al., 1992; Werman et al., 2003; Komprda,
2012; Kagan et al., 2014; Bendimerad-Benmokhtar
et al., 2017).
Serum enzymes (ALT, AST) in the current study
had doubled values in G2-Ps in comparison to G1-
Ng, reecting the dangerous eect of the challenge
on the liver. Over the course of the experiment, the
serum levels of ALT and AST were reduced in
N. oculata treated birds, indicating its protective
eects on the liver cells. These findings are in agree-
ment with previous research (Bhattacharyya &
Mehta, 2012; Nacer et al., 2020; Abd El-Hamid et al.,
2022; El-Hawy et al., 2022). Serum urea readings
were reduced after N. oculata intake in the current
work, which is indicative of a beneficial eect on the
kidneys as stated previously (Nuño et al., 2013;
Aboulthana et al., 2018; Nacer et al., 2020).
In this study, G1-Ng showed normal caecal histo-
logical architecture as reported by Cukrowska et al.
(2017) and Clark and Mach (2017). The ultrastructure
of the G2-Ps cecal wall revealed histopathological
damage, that was consistent with previous studies
(Teshfam & Rahbari, 2003; Dai et al., 2005; Bashtar
et al., 2010). This damage could be attributed to proto-
zoan-induced mechanical irritation (Singla et al.,
2000). The G3-Clo showed a lower number of cocci-
dian developing stages in comparison to G2-Ps.
These observations could be attributed to the ability
of clopidol to destroy some enterocytes which, in
turn, decrease its coccidiostatic activity (Hafeez et al.,
2022), also clopidol could increase cell death and
rates of Eimeria infection of enterocytes (Picard et al.,
2013). There was a significant decrease in the number
of developmental stages of Eimeria spp. and degenerated
stages, as well as a normal mitochondrial appearance
with mild or no cellular structural damage in G4-NOa
and G5-NOb. Similar findings were recorded by Cere-
zuela et al. (2012). The ability of N. oculata to improve
enterocyte performance and remarkably decline Eimeria
developmental stages could be attributed to its ability to
increase the number of intestinal macrophages (Reyes-
Becerril et al., 2013; Levine et al., 2018).
E. tenella aldolase is an essential enzyme for
Eimeria energy metabolism (Hu et al., 2022).
EtAMA1 and EtMIC3 were expressed at high levels
in the sporozoite stage, facilitating their invasion
into host cells (Jiang et al., 2012; Chen et al., 2021).
In the current study, molecular docking revealed a
higher binding anity of N. oculata for E. tenella aldo-
lase, EtAMA1, and EtMIC3, which hindered glucose
metabolism, host cell adhesion, and invasion of
Eimeria. Therefore, E. tenella aldolase, EtAMA1, and
EtMIC3 have been considered potential drug targets.
In conclusion, administering 0.2% dried N. oculata
powder in the diet of broilers challenged with 5 × 10
3
sporulated oocysts of Eimeria spp. from 1 day old had
eects that were close to or better than those of clopi-
dol in terms of improving clinical parameters, meat
quality, clinicopathological parameters, and histologi-
cal parameters. We recommend carrying out extensive
experiments under field conditions with higher doses
of oocyst challenge.
Acknowledgements
The authors would like to expresses sincere appreciation to
Ass. Prof.: Mohamed Elbadawy (Department of Pharma-
cology, Faculty of Veterinary Medicine, Benha University),
for his valuable support during the review and editing of
the paper.
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
The authors did not receive support from any organization
for the submitted work.
ORCID
Marwa I. Abdel Haleem http://orcid.org/0000-0003-
3161-661X
Shimaa N. Edris http://orcid.org/0000-0002-1703-0981
Hanan A.A. Taie http://orcid.org/0000-0002-2497-3611
Samah M. Abdel Gawad http://orcid.org/0000-0001-
7027-9738
Sawsan S. Elbasuni http://orcid.org/0000-0002-2393-
9948
AVIAN PATHOLOGY 15
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