Nutrición Animal Tropical 19 (2): 61-82 Julio-Diciembre, 2025
ISSN: 2215-3527 / DOI: 10.15517/j6gfv014
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____________________________________________________________________________________________________________________
1 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
aoayeni@futa.edu.ng (https://orcid.org/0000-0001-7558-0781).
2 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
madegbenro@futa.edu.ng (https://orcid.org/0000-0001-9275-3998).
3 Federal College of Agriculture, Department of Animal Health and Production Technology. Akure, Nigeria. Autor para
correspondencia: oluwafemijoshua333@gmail.com (https://orcid.org/0009-0000-2838-215X).
4 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
oladayoto@futa.edu.ng (https://orcid.org/0000-0003-0549-8613).
5 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
ebunoluwa380@gmail.com (https://orcid.org/0009-0005-7856-0176).
6 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
olabanjioluwatoyin3@gmail.com (https://orcid.org/0009-0008-7944-6617).
7 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
olasunkanmimutairu8@gmail.com (https://orcid.org/0009-0004-5768-0155).
8 Federal University of Technology, Department of Animal Production and Health. Akure, Nigeria. Correo electrónico:
ooagbede@futa.edu.ng (https://orcid.org/0000-0001-7297-6350).
Recibido: 15 marzo 2024 Aceptado: 08 septiembre 2025
Esta obra está bajo licencia internacional CreativeCommons Reconocimiento-NoComercial-SinObrasDerivadas 4.0.
Scientific article
Haemato-biochemical indices and antioxidant status of layers-fed diets supplemented
with garlic-composite leaf mix
Akinlolu Oluwafemi-Ayeni
1, Muyiwa Adegbenro2, Joshua Oluwafemi-Olaleye3, Taiwo Olarotimi-Oladayo4,
Florence Ebunoluwa Ajayi5, Oluwatoyin Olabanji6, Mutairu Olasunkanmi7, Johnson Oluwasola-Agbede8
ABSTRACT
The study aimed to evaluate haemato-biochemical indices and antioxidant status in laying hens
fed varying levels of garlic-composite leaf mix (GCM), addressing limited research in this area. A
twelve-week feeding trial was conducted at the Poultry unit of the Teaching and Research Farm
(TRF) of the Federal University of Technology, Akure (FUTA), in Nigeria. The soursop, avocado,
sandpaper, and gliricidia leaves were manually harvested, air-dried, and milled to produce the
GCM; and then combined with garlic in a 2:2:2:3:1 ratio, respectively. The mix was added to the
basal diet, designated as diet 1 (control, 0 g/kg), diet 2 (5 g/kg), diet 3 (10 g/kg), diet 4 (15 g/kg),
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and diet 5 (20 g/kg). A total of 135 hens were distributed across these 5 dietary treatments with
3 replicates, each containing 9 birds, in a completely randomized design. Packed cell volume, red
blood cell count, and haemoglobin concentration showed significant differences (p < 0.05),
whereas other haematological parameters did not (p > 0.05). Mean cell haemoglobin
concentration presented higher values observed in the GCM-fed groups (32.45 ± 0.51 - 32.74 ±
0.34 g/dl) compared to the control diet (32.13 ± 0.07 g/dl). White blood cell counts were highest
(6.60 ± 1.27×106/L) in birds fed diet 4. Alanine aminotransferase levels were lowest (10.20 ± 2.31
IU/L) in birds fed diet 3 and highest (14.40 ± 0.35) in those supplemented with diet 5. Superoxide
dismutase and glutathione peroxidase levels increased significantly (p < 0.05) in birds fed the test
diets. Catalase levels were higher (78.56 ± 11.74) in birds on the control diet than those on test
diets (20.46 ± 2.13 - 56.86 ± 19.78). The study concluded that the inclusion of GCM had no adverse
effects on the health of laying hens but rather helped maintained their haematological, blood
serum, and antioxidant parameters.
Keywords: haematology, serum biochemistry, antioxidant, Isa brown, garlic mix, composite leaf
mix.
RESUMEN
Índices hemato-bioquímicos y estado antioxidante en aves ponedoras alimentadas con dietas
suplementadas con una mezcla de hojas compuestas con ajo. El estudio tuvo como objetivo
evaluar los índices hemato-bioquímicos y el estado antioxidante en gallinas ponedoras
alimentadas con diferentes niveles de una mezcla de hojas compuestas con ajo (MCA),
abordando la investigación limitada en esta área. Se realizó un ensayo de alimentación de 12
semanas en la Unidad Avícola de la Granja de Enseñanza e Investigación (TRF) de la Universidad
Federal de Tecnología de Akure (FUTA), Nigeria. Las hojas de guanábana, aguacate, higuera de
lija y gliricidia fueron cosechadas manualmente, secadas al aire y molidas para producir la MCA;
se combinaron con el ajo en una proporción de 2:2:2:3:1, respectivamente. La mezcla se añadió
a la dieta base, designadas como dieta 1 (control, 0 g/kg), dieta 2 (5 g/kg), dieta 3 (10 g/kg), dieta
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
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4 (15 g/kg) y dieta 5 (20 g/kg). Un total de 135 gallinas fueron distribuidas en 5 tratamientos con
3 repeticiones, cada una con 9 aves, en un diseño completamente aleatorizado. El volumen
celular empaquetado, eritrocitos y la hemoglobina fueron significativos (p < 0.05), mientras que
otros parámetros hematológicos no fueron significativos (p > 0.05). La concentración media de
hemoglobina celular presentó valores más altos observados en grupos alimentados con MCA
(32.45 ± 0.51 - 32.74 ± 0.34 g/dL) en comparación con la dieta control (32.13 ± 0.07 g/dL). El
conteo de glóbulos blancos fue mayor (6.60±1.27 ×106/L) en las aves alimentadas con la dieta 4.
Los niveles de alanina aminotransferasa fueron más bajos (10.20 ± 2.31 IU/L) en las aves
alimentadas con la dieta 3 y más altos (14.40 ± 0.35 IU/L) en aquellas suplementadas con la dieta
5. El superóxido dismutasa y la glutatión peroxidasa incrementaron significativamente (p < 0.05)
en las aves alimentadas con dietas experimentales. Los niveles de catalasa fueron más altos (78.56
± 11.74) en las aves de la dieta control que en aquellas de las dietas de prueba (20.46 ± 2.13-
56.86 ± 19.78). El estudio concluyó que el MCA no tuvo efectos adversos en la salud de las gallinas
ponedoras, sino que mantuvo sus parámetros hematológicos, bioquímicos y estado oxidativo.
Palabras clave: hematología, bioquímica sérica, antioxidante, isa brown, mezcla de ajo, mezcla
de hojas compuestas.
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INTRODUCTION
The increasing prevalence of antibiotic-resistant organisms has prompted extensive research into
reliable alternatives, leading to the identification of plant-derived products such as leaves and
seeds. In addition to the inherent benefits of natural plant extracts, recent studies have shown
that herbal medicines and their derivatives can positively influence laying performance and
enhance the antioxidant capacity of poultry (Alagawany et al., 2017).
In this context, phytogenic feed additives, particularly those derived from plants, have emerged
as potential alternatives to antibiotic growth promoters in poultry nutrition and other areas of
livestock production. Given the growing global concerns over meat and egg safety, the search
for effective antibiotic substitutes is more urgent than ever. Among these alternatives, plant
leaves and their extracts have recently been recognized as some of the most promising options
(Mahrose et al., 2019).
Research has revealed that certain types of plant foliage contain chemical compounds with
potent medicinal properties, which can improve reproductive tract function, enhance overall
animal welfare, and elevate the quality of animal-derived products such as meat and eggs
(Jamshidparvar et al., 2017). Moreover, some bioactive compounds found in plant-based
products have demonstrated a significant prophylactic role in preventing various infections
(Hashemi and Davoodi, 2012).
The inclusion of herbal medicine in poultry feed has been shown to stimulate feed intake, thereby
enhancing and sustaining bird welfare and productivity (Windisch et al., 2008). The use of plant
foliage in poultry farming not only contributes to antimicrobial effects against pathogenic
microorganisms but also promotes improved nutrient utilization in the gut (Dhama et al., 2015).
Based on this evidence, it is proposed that plant leaves possess significant phytogenic activity
and can be effectively used in layer management to improve performance and health.
Hematological and serum biochemical parameters are often influenced by factors such as
medication, toxic substances, diet, infections, age, and sex of the birds (Schmidt et al., 2009). For
instance, dietary supplementation with soursop leaves has been found to reduce intramuscular
lipid cell muscle fibers, carcass yield, and carcass components. Avocado leaves have
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
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demonstrated therapeutic potential for treating conditions such as anemia and hypertension and
exhibit notable antioxidant properties. Similarly, sandpaper leaf supplementation has been shown
to reduce free fatty acids, acid value, and peroxide value. Additionally, garlic has gained a
longstanding reputation across various cultures as both a prophylactic and therapeutic medicinal
plant (Huyghebaert, 2003).
However, research on the use of soursop (
Annona muricata
), avocado (
Persea americana
),
sandpaper (
Ficus exasperata
), and gliricidia (
Gliricidia sepium
) leaves—whether individually or in
combination—as dietary supplements to enhance the health of laying hens remains limited. This
gap in knowledge forms the basis of the present study, which was designed to investigate the
hematological and biochemical indices, as well as the antioxidant status, of laying hens fed a
composite mixture of soursop leaf, avocado leaf, sandpaper leaf, gliricidia leaf, and garlic powder.
MATERIALS AND METHODS
Experimental Site
This research was conducted at the Poultry Unit of the Teaching and Research Farm (TRF) of the
Federal University of Technology, Akure (FUTA), Nigeria. Akure is located at latitude 7.3070°N
and longitude 5.1398°E, within the humid rainforest zone of western Nigeria, which is
characterized by bimodal rainfall patterns and high humidity during the rainy season (Agbelade
and Akindele, 2013). Additional laboratory analyses were performed at the Central Research
Laboratory of FUTA.
The birds were housed in a three-tier Californian colony cage system [dimensions: 6 m (length)
× 4 m (width) × 3 m (height)], with three birds per cell, three cage units per replicate, and three
replicates per treatment. The cages were equipped with open galvanized feeders and plastic pipe
drip water troughs. The average annual temperature and relative humidity in the poultry house
was 27 °C and 75%, respectively (Olabode and Adeleke, 2017).
Each bird received 110 g of feed per day, divided into two portions administered at 9:00 a.m. and
4:00 p.m. Water was provided
ad libitum
.
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Test ingredient preparation
Fresh soursop, avocado, sandpaper, and gliricidia leaves were harvested from mature stems. The
leaves were chopped and air-dried at 35 °C until reaching average moisture contents of 11.01%
(soursop), 10.45% (avocado), 11.61% (sandpaper), and 13.5% (gliricidia). Once dried, the leaves
were ground into a fine powder with a particle size of 0.02 mm using a Granulex® 3 Series
hammer mill. Garlic bulbs were obtained from a local vendor and similarly processed into powder.
Subsequently, the leaf meals (soursop, avocado, sandpaper, and gliricidia) and garlic powder
were blended in a ratio of 2:2:2:3:1, respectively, to produce a garlic-composite leaf meal (GCM).
Antioxidant concentration and activity were determined using a spectrophotometric method.
Four replicates of each test ingredient were analyzed and labeled S1, S2, S3, S4, and S5. Samples
were stored in well-covered Petri dishes before analysis.
Experimental diets
The gross composition of the experimental diets is presented in Table 1, and Table 2 provides the
calculated nutrient composition.
The basal diet was formulated to contain 17% crude protein (CP) and 2655 kcal/kg metabolizable
energy (ME), following the nutritional requirements of laying hens at 19 weeks of age, as
recommended by the NRC (1994). Based on this formulation, five experimental diets were
developed. Diet 1 served as the control and did not include any supplementation. Diet 2 included
125 g of GCM per 25 kg of feed (5 g/kg), while Diet 3 contained 250 g per 25 kg (10 g/kg). Diets
4 and 5 were supplemented with 375 g (15 g/kg) and 500 g (20 g/kg) of GCM per 25 kg of feed,
respectively. In all supplemented treatments, the GCM was thoroughly mixed into the feed
offered to the birds.
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
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Table 1. Gross composition of the experimental basal diet.
Ingredients
Quantity (%)
Maize
55.45
Soybean meal
9.00
Groundnut cake
16.50
Wheat offal
6.50
Dicalcium phosphate
1.50
Fishmeal
0.50
Limestone
9.70
Lysine
0.15
Methionine
0.15
Salt
0.30
Premix
0.25
Total
100.00
Table 2. Calculated analysis of the experimental basal diet.
Nutrient
Value
Metabolizable energy (kcal/kg)
2655.00
Crude protein (%)
17.00
Moisture content (%)
13.00
Crude fiber (%)
3.80
Calcium (%)
4.01
Available phosphorus (%)
0.64
Methionine (%)
0.41
Lysine (%)
0.84
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Experimental birds and management
A total of 135 Isa Brown laying hens, 19 weeks of age and at the point of lay, were used in this
experiment. The birds were randomly assigned to one of five dietary treatment groups
corresponding to 0, 5, 10, 15, and 20 g/kg of garlic-composite leaf meal (GCM). The treatments
reflected different supplementation levels of the GCM, and the birds were fed their respective
diets for a period of 12 weeks. Each treatment was replicated three times, with nine birds per
replicate, in a completely randomized design. Replicates were tagged as Diets 1 to 5.
Biochemical analysis and data collection
Three laying birds per treatment were fasted prior to slaughter and exsanguination. Blood
samples were collected from the jugular vein into three labeled, heparinized tubes. Additional
blood samples were collected into clean, dry centrifuge tubes without anticoagulants and
transported to the laboratory for analysis of hematological indices, serum biochemical
parameters, and antioxidant enzyme activities. Analyses were performed using an automated
hematology analyzer (BIOBASE, BK-6400).
Hematological parameters measured included packed cell volume (PCV), erythrocyte count,
hemoglobin concentration, mean corpuscular volume (MCV), and mean corpuscular hemoglobin
(MCH). Serum biochemical indices determined were total protein, albumin, globulin, alanine
aminotransferase (ALT), aspartate aminotransferase (AST), serum cholesterol, and creatinine
levels.
Antioxidant enzyme concentrations measured included glutathione peroxidase (GSH-Px),
superoxide dismutase (SOD), and catalase (CAT). Serum GSH-Px activity was determined
according to the method described by Weiss et al. (1980), using Ransel (RANDOX/RS-504; Randox
Laboratories, Crumlin, UK). SOD activity was measured following the procedure of Nishikimi et
al. (1972), using a commercial enzyme kit (Ransod, RANDOX/SD-125; Randox Laboratories,
Crumlin, UK).
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Statistical analysis
The statistical model used to analyze the generated data was an inferential statistical model. All
data collected were subjected to analysis of variance (ANOVA) using SPSS software, version 25.
Duncan’s Multiple Range Test (DMRT, 2002) was employed to separate means when significant
differences were observed. Below is the statistical model:
Yij=µ+Ti +Eij
Where
Yij is the individual observation of all treatments
µ is the universal means
Ti is the effect of the diets i.e, treatments
Eij is the experimental error
RESULTS
Antioxidant properties of garlic-leaf meals and the composite mix
Table 3 presents the antioxidant compositions of soursop, avocado, sandpaper, gliricidia leaf
meal, garlic mix, and composite leaf mix. The results indicate that GCM exhibited mean values of
7.93 mg/100 g, 18.31%, 51.26 mg/100 g, and 0.23 mg/100g for flavonoids, 2,2-Diphenyl-1-
picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP), and phenol content,
respectively, with corresponding coefficient variations of 72.07%, 73.54%, 58.91%, and 31.89%.
Soursop leaf meal contained the highest flavonoids content (14.17 mg/100 g) as compared to
gliricidia, which recorded the lowest (0.62 mg/100 g). DDPH was, however, higher for avocado
leaf meal (33.28%), while the lowest (5.45%) was recorded in sandpaper leaf meal.
The phenol content was highest (0.35 mg/100 g) in avocado leaf meal and lowest (0.14 mg/100
g) in garlic meal.
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Table 3. Antioxidant composition of leaf meals and composite leaf mix meal (GCM).
Parameters
Antioxidants
SSM
AVM
SPM
GCM
GLM
CLM
Mean
Stdev
CoV
Flavonoids
(mg/100g)
14.17
12.30
4.12
0.62
12.49
3.87
7.93
5.71
72.07
DPPH (%)
25.31
33.28
5.45
7.55
5.88
32.35
18.31
13.46
73.54
FRAP
(mg/100g)
80.15
80.02
29.11
15.10
28.14
75.01
51.26
30.19
58.91
Phenol
(mg/100g)
0.25
0.35
0.21
0.14
0.22
0.18
0.23
0.07
31.89
SSM: Soursop leaf meal; AVM: Avocado leaf; SPM: Sandpaper leaf; GCM: Gliricidia leaf meal; GLM: Garlic
meal; CLM: Composite leaf mix; Stdev: Standard error of variation; CoV: Coefficient of variation; DDPH: 2,2-
Diphenyl-1-picrylhydrazyl; FRAP: ferric ion reducing antioxidant power.
The haematological parameters of laying hens fed diets supplemented with GCM
The hematological parameters of laying hens fed diets containing varying levels of garlic-
composite leaf meal (GCM) are presented in Table 4. The results showed that among all the
hematological parameters evaluated, only packed cell volume (PCV), red blood cell count (RBC),
and mean corpuscular volume (MCV) were significantly affected by the dietary treatments (p <
0.05), whereas mean corpuscular hemoglobin (MCH), granulocyte count, and lymphocyte count
were not significantly influenced (p > 0.05).
The highest PCV (38.50 ± 0.29%) was observed in birds fed Diet 3, compared to those on other
treatments. Similarly, RBC count was highest in birds fed Diet 3 (5.05 ± 0.38 × 10⁶/L), while the
lowest value (2.95 ± 0.03 × 10⁶/L) was recorded in birds fed Diet 5. The highest MCV (93.22 ±
0.07 fL) was recorded in birds fed Diet 5, whereas the lowest (58.11 ± 0.13 fL) was found in birds
fed Diet 4.
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Although not statistically significant, MCH was highest in birds fed Diet 5 (31.07 ± 0.02 pg/cell)
and lowest in those on Diet 4 (19.37 ± 0.04 pg/cell). Hemoglobin concentration peaked in birds
fed Diet 3 (12.83 ± 0.10 g/dL) and was lowest in those on Diet 4 (7.17 ± 0.10 g/dL). The highest
lymphocyte percentage (67.00 ± 2.89%) was recorded in birds fed Diet 2, while the lowest (52.00
± 6.93%) was found in birds on Diet 5. Finally, the highest granulocyte percentage (48.00 ± 6.93%)
was observed in birds fed Diet 5, and the lowest (31.00 ± 4.04%) in those fed Diet 2.
Table 4. Hematology parameters of laying hens fed diets containing varying levels of garlic-
composite mix supplementary.
Treatment
Diet 1
(Control)
Diet 2
(5 g/kg)
Diet 3
(10 g/kg)
Diet 4
(15 g/kg)
Diet 5
(20 g/kg)
P-Value
PCV %
28.00 ± 1.73b
30.50 ± 1.44b
38.50 ± 0.29a
21.50 ± 0.29c
27.50 ±
0.29b
<0.001*
RBC (x106/L)
3.40 ± 0.35bc
4.15 ± 0.49ab
5.05 ± 0.38a
3.70 ± 0.06bc
2.95 ± 0.03c
0.008*
MCHC (g/dL)
32.13 ± 0.07
32.74 ± 0.34
32.45 ± 0.51
32.57 ± 0.44
32.74 ± 0.34
0.762
MCV (fL)
86.61 ± 13.92
75.42 ± 5.44
77.65 ± 6.34
58.11 ± 0.13
93.22 ± 0.07
0.054*
MCH (pg/cell)
28.87 ± 4.64
25.14 ± 1.81
25.88 ± 2.12
19.37 ± 0.04
31.07 ± 0.02
0.054*
Haemoglobin
(Hb g/dL)
9.33 ± 0.58b
10.17 ± 0.48b
12.83 ± 0.10a
7.17 ± 0.10c
9.17 ± 0.10b
<0.001*
WBC (x 106/L)
3.20 ± 0.58
5.90 ± 2.25
2.90 ± 0.06
6.60 ± 1.27
4.20 ± 0.35
0.192
Granulocyte (%)
36.00 ± 2.31
31.00 ± 4.04
42.00 ± 1.15
42.00 ± 0.00
48.00 ± 6.93
0.074
Lymphocyte (%)
64.00 ± 2.31
67.00 ± 2.89
58.00 ± 1.15
57.00 ± 0.58a
52.00 ± 6.93
0.086
Monocytes (%)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.00 ± 0.58
0.00 ± 0.00
0.072
a, b, and c: Means on the rows with different superscripts are significantly different.
PCV: packed cell volume; RBC: red blood cell; MCHC: mean cell haemoglobin concentration; MCV: mean
cell volume; MCH: mean cell haemoglobin; WBC: white blood cell.
*Significant at the 0.05 probability level.
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Serum biochemical indices of laying hens fed diet supplemented with GCM
The serum biochemical indices of laying hens fed diets containing varying levels of GCM, as
presented in Table 5, revealed that among all parameters measured, aspartate aminotransferase,
cholesterol, creatinine, total protein, albumin, and globulin were significantly (p < 0.05) influenced
by the dietary treatments. It was also observed that globulin levels increased in the treated birds,
which resulted in an increase in total protein production in these groups. Birds fed diet 3 recorded
the highest aspartate aminotransferase and cholesterol levels (78.70 ± 5.95 IU/L and 3.95 ± 0.26
mmol/L, respectively) compared to birds fed diet 1 and diet 5. Creatinine levels were higher (79.85
± 12.27) in birds fed diet 5 compared to those fed the other treatments.
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Table 5. Serum biochemistry indices of laying hens fed diets containing varying levels of garlic-
composite mix supplementary.
Treatment
Diet 1
(Control)
Diet 2
(5 g/kg)
Diet 3
(10 g/kg)
Diet 4
(15 g/kg)
Diet 5
(20 g/kg)
P-Value
AST (IU/L)
52.45 ±
0.09c
62.75 ± 3.44b
78.70 ± 5.95a
66.45 ± 0.72b
71.85 ± 2.22a
0.002*
ALT (IU/L)
10.55 ± 0.32
13.35 4± 0.38
10.20 ± 2.31
12.20 ± 1.15
14.40 ± 0.35
0.131
Cholesterol(mmol/L)
3.45 ± 0.03b
3.55 ± 0.09b
3.95 ± 0.26a
3.20 ± 0.06b
3.20 ± 0.00b
0.010*
Creatinine (mmol/L)
39.90 ±
4.62bc
53.20 ±
12.30b
18.65 ± 1.53c
21.30 ± 3.06c
79.85 ±
12.27a
0.002*
Total protein (g/L)
59.75 ±
0.32c
73.65 ± 1.18a
70.40 ±
2.42ab
68.40 ± 1.56b
67.70 ±
0.52b
0.001*
Albumin (g/L)
45.35 ±
1.36a
37.55 ±
1.59bc
39.65 ±
2.68bc
36.65 ± 0.95c
42.55 ±
1.36ab
0.024*
Globulin (g/L)
14.40 ±
1.04d
36.10 ± 0.40a
30.75 ± 0.26b
31.75 ±
2.51ab
25.15 ± 1.88c
<0.001*
Glucose (mg/dL)
217.50 ±
1.44
208.50 ±
15.88
196.50 ±
48.21
219.00 ± 4.04
208.50 ±
2.60
0.956
a, b, and c: Means on the rows with different superscripts are significantly different.
AST: Aspartate Amino Transferase; ALT: Alanine Amino Transferase.
*Means significant difference.
Antioxidant properties of garlic-composite leaf supplemented in the diet of laying hens
Table 6 shows the antioxidant properties of the diets evaluated, containing varying levels of GCM.
From the results of all the parameters measured, only SOD was significantly (p < 0.05) influenced
by the dietary treatments. The highest SOD activity (90.73 ± 1.97 U/mL) was observed in laying
hens fed diet 4, while the lowest (59.02 ± 2.25 U/mL) was recorded in hens fed diet 1. CAT and
GSH levels were not significantly (p > 0.05) influenced by the dietary treatments.
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Table 6. Antioxidant parameters of the diets evaluated containing varying levels of garlic-
composite leaf mix supplementary.
Treatment
(U/mL)
Diet 1
(Control)
Diet 2
(5 g/kg)
Diet 3
(10 g/kg)
Diet 4
(15 g/kg)
Diet 5
(20 g/kg)
P-
Value
SOD
59.02 ± 2.25d
74.63 ± 4.51bc
87.32 ±
2.82ab
90.73 ± 1.97a
64.39 ±
7.04cd
0.001*
CAT
78.56 ± 11.74
56.86 ± 19.78
20.46 ± 2.13
47.96 ± 23.03
23.53 ± 3.90
0.092
GSH
215.09 ± 16.60
236.84 ± 2.60
223.34 ± 0.29
224.84 ± 5.77
232.84 ± 5.20
0.426
a, b, c, and d: Means on the rows with different superscripts are significantly different.
SOD: superoxide dismutase; CAT: catalase; and GSH: glutathione peroxidase.
*Means significant difference.
DISCUSSION
The results of the haematological indices showed that the packed cell volume (PCV) of birds
across the treatments ranged from 21.50% to 38.50%, with no discernible trend. According to
Akanbi et al. (2020), the normal PCV range for adult chickens is 30-40%. PCV serves as a useful
indicator of nutritional anaemia and general physiological status. Elevated PCV may indicate
dehydration or stress, while a low PCV suggests anaemia or nutritional deficiencies. The low PCV
values recorded in birds fed Diets 1, 4, and 5 may be attributed to iron or vitamin deficiencies or
excessive water intake, possibly due to high ambient temperatures (45 °C). However, as birds fed
Diets 2 and 3 presented PCV values within the normal range, it is unlikely that the GCM
supplementation had a negative influence on PCV. Therefore, GCM inclusion at the tested levels
can be considered physiologically safe for PCV maintenance.
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Regarding RBC, the values recorded ranged from 2.95 to 5.05 × 10⁶ µL, aligning with the normal
range (2.5–3.5 × 10⁶ µL) reported by Bounous and Stedman (2000). Birds fed Diets 2 and 3
exhibited higher RBC counts, which may not be directly linked to GCM supplementation, but
instead to other physiological responses, such as dehydration, as confirmed during the post-
mortem examination of control birds. Given the role of RBCs in oxygen transport, elevated counts
may reflect compensatory mechanisms in response to physiological stress.
Haemoglobin concentrations ranged between 7.17 and 12.83 g/dL, mostly within the
physiological range of 9–13 g/dL (Akanbi et al., 2020). Although some values were slightly below
this threshold, particularly in birds fed Diet 4, the variations remained within acceptable biological
limits, suggesting no detrimental effects from GCM (Akanbi et al., 2020).
White blood cell (WBC) counts, and differential indices such as lymphocytes and granulocytes
were not significantly influenced by the dietary treatments, indicating that GCM supplementation
did not negatively impact the birds’ immune status (Schmidt et al., 2009).
Mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) were significantly
affected by dietary GCM levels, while mean corpuscular haemoglobin concentration (MCHC)
remained unaffected. The values for MCV (80–100 fL), MCH (27–33 pg/cell), and MCHC (32–36
g/dL) fell within normal physiological ranges (Akanbi et al., 2020). These indices are crucial for
assessing red blood cell morphology and function. Elevated MCH and MCV, coupled with normal
MCHC, suggest the absence of macrocytic anaemia or dietary toxicity (Roberts et al., 2000; Etim
et al., 2014). This reinforces the notion that GCM did not introduce toxic elements into the feed.
In terms of serum biochemistry, GCM appeared to reduce albumin levels at lower inclusion rates
but increased it at the highest supplementation level (Diet 5). This contrasts with the findings of
El-Katcha et al. (2016), who reported that garlic supplementation at 50 mg/kg improved albumin
content. Similarly, other studies found no significant influence of garlic extract on total protein
and albumin levels compared to controls (El-Katcha et al., 2016).
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Serum cholesterol levels initially rose with GCM inclusion but declined in birds fed Diets 4 and 5.
This agrees with Elbaz et al. (2021), who noted that garlic at 20 g/kg significantly reduced serum
cholesterol in broilers. Several other studies (Horton et al., 1991, Khan et al., 2008; Rehman et al.,
2011; Khan et al., 2012; Omer et al., 2019) have similarly shown that garlic-based supplements
improve lipid profiles. In this study, birds fed Diets 4 and 5 exhibited a 7.25% reduction in total
cholesterol compared to controls, corroborating previous findings on garlic’s
hypocholesterolemic properties (El-Katcha et al., 2016).
Total serum protein and globulin levels are critical indicators of immune function. In this study,
globulin was elevated in birds fed Diet 2, possibly explaining the lower mortality in this group.
Albumin, essential for nutrient transport and tissue repair, also plays a role in binding free radicals,
suggesting potential antioxidant benefits of GCM (Piotrowska et al., 2021; Oleforuh-Okoleh et al.,
2015). These findings align with Oleforuh-Okoleh et al. (2015), who reported improved serum
biochemistry in broilers supplemented with garlic and ginger.
Alanine aminotransferase (ALT) levels were highest in birds fed Diet 3. ALT is primarily a hepatic
enzyme involved in amino acid metabolism and detoxification (Kim et al., 2008). Although
elevated ALT may suggest liver stress, it is also influenced by factors such as glucose metabolism
(Kim et al., 2008). In this study, the increase in ALT may be linked to higher glucose concentrations
rather than hepatocellular damage.
The antioxidant enzymes evaluated, glutathione peroxidase (GSH), superoxide dismutase (SOD),
and catalase (CAT), provide insight into the birds’ oxidative status. SOD levels increased
significantly with GCM supplementation, while CAT decreased. SOD neutralizes superoxide
radicals, converting them into hydrogen peroxide, which is then broken down by CAT (Ighodaro
and Akinloye, 2018; Nandi et al., 2019). The imbalance observed (elevated SOD and reduced CAT)
may be a result of environmental stressors such as high ambient temperatures or humidity.
Elevated SOD has been associated with neurological disorders and apoptosis (Younus, 2018) and
may explain the gliosis observed in the brain cortex of GCM-fed birds. However, the concurrent
increase in GSH activity suggests a compensatory antioxidant response (Yoon et al., 2016).
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
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Glutathione plays a crucial role in cellular defense against oxidative stress. Increased GSH activity,
as observed in GCM-fed birds, supports the hypothesis that the phytogenic additives in the GCM
mixture, soursop, avocado, sandpaper, and gliricidia leaves, contributed to enhanced antioxidant
capacity (Sameer, 2013). This aligns with findings from Yoon et al. (2016), who linked elevated
GSH levels to improved health and resistance to infection.
In summary, GCM supplementation at levels up to 20 g/kg in the diets of laying hens had no
adverse effects on haematological or serum biochemical indices and even conferred potential
health benefits, particularly regarding antioxidant status and cholesterol reduction.
CONCLUSION
Based on these findings, the addition or supplementation of garlic composite leaf mix (soursop,
avocado, sandpaper and gliricidia) may have a positive effect on the birds’ biomarkers, although
without a consistent pattern. This supplementation could improve the immune response and
cholesterol levels of the laying hens, thereby enhancing their overall performance. Incorporating
composite mixtures into the basal diet of laying hens may contribute to healthier Isa Brown
chickens and offer an opportunity to improve productivity. Furthermore, it is recommended that
future research be conducted to investigate the potential synergistic effects that GCM on the
health status and egg production performance of laying hens.
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LITERATURE CITED
Agbelade, A. D. and Akindele, S. O. (2013). Land use mapping and tree species diversity of Federal
University of technology, Akure. American International Journal of Contemporary
Research, 3 (2), 104-113.
Akanbi, O. M., Aigbogun, G. O. and Oluponna, J. A. (2020). Effect of Ginger (
Zingiber officinale
)
and garlic (
Allium sativum
) on the performance, haematology and serum biochemical
indices of laying hens. Anesthesia and Surgery Open Access Journal, 1 (3), 1-5.
Alagawany, M., Abd El-Hack, M. E., Raqab, M. and Marappan, F. (2017). Rosmarinic acid: Modes
of action, medicinal values and health benefits. Animal Health Research Reviews, 18 (2),
167-176. http://dx.doi.org/ 10.1017/S1466252317000081.
Bounous, D. and Stedman, N. (2000). Normal avian haematology: chicken and turkey. In: Schalm’s
Veterinary haematology. Feldman BF, Zinkl J.G, Jain NC (eds). New York, Wiley, 1147-1154
pp.
Dhama, K., Lateef, S. K., Mani, S., Samad, H. A., Karthik, K., Tiwari, R. and Tufarelli, V. (2015). Multiple
beneficial applications and modes of action of herbs in poultry health and production- A
review. International Journal of Pharmacology, 11, 152-17. http://dx.doi.org/
10.3923/ijp.2015.152.176.
Elbaz, A. M., Ibrahim, N. S., Shehata, A. M., Mohamed, N. G. and Abdel-Moneim, A. M. E. (2021).
Impact of multi-strain probiotic, citric acid, garlic powder or their combinations on
performance, ileal histomorphometry, microbial enumeration and hormonal immunity of
broiler chickens. Tropical Animal Health and Production, 53, 1-10.
http://dx.doi.org/10.1007/s11250-021-02554-0.
El-katcha, M. I., Soltan, M. A., Sharaf, M. M. and Hasen, A. (2016). Growth performance, immune
response, blood serum parameters, nutrient digestibility and carcass traits of broiler
chicken as affected by dietary supplementation of garlic extract (
Allicin
). Alexandria
Journal of Veterinary Sciences, 49 (2), 50-64. http://dx.doi.org/10.5455/ajvs.219261.
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
_________________________________________________________________________________________________________________
________________________________________________________________________________________________
Nutrición Animal Tropical 19 (2): 63-84. ISSN: 2215-3527 / 2025
79
Etim, N. N., Williams, M. E., Akpabio, U. and Offiong, E. E. A. (2014). Haematological parameters
and factors affecting their value. Agricultural Science, 2, 37-47. http://dx.doi.org/
10.12735/as.v2i1p37.
Hashemi, S. R. and Davoodi, H. (2012). Herbal plants as new immunostimulator in poultry industry:
A review. Asian Journal of Animal and Veterinary Advances, 7, 105-116. http://dx.doi.org/
10.3923/ajava.2012.105.116.
Horton, P., Ruban, A. V., Rees, D., Pascal, A. A., Noctor, G. and Young, A. J. (1991). Control of the
light-harvesting function of chloroplast membranes by aggregation of the LHCII
chlorophyll-protein complex. FEBS letters, 292 (1-2), 1-4. https://doi.org/ 10.1016/0014-
5793(91)80819-o.
Huyghebaert, G. (2003). Replacement of antibiotics in poultry. Proceedings of the Eastern
Nutrition Conference, 2003, Quebec: Canada, Quebec: UON; 2003.
Ighodaro, O. M. and Akinloye, O. A. (2018). First line defence antioxidants-superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidise (GPX): Their fundamental role in the
entire antioxidant defence grid. Alexandria journal of medicine, 54 (4), 287-293.
https://doi.org/10.1016/j.ajme.2017.09.001.
Jamshidparvar, A., Javandel, F., Seidavi, A., Pena, F., L., Martinez, A., Aviles, C., Aguera, E. and
Núñez-Sánchez, N. (2017). Effects of golpar (
Heracleum persicum
Desf.) and probiotics in
drinking water on performance, carcass characteristics, organ weights, blood plasma
constituents, and immunity of broilers. Environmental Science and Pollution Research, 24,
23571-23577. https://doi.org/10.1007/s11356-017-9983-4.
Khan, S., Cao, Q., Zheng, Y. M., Huang, Y. Z. and Zhu, Y. G. (2008). Health risks of heavy metals in
contaminated soils and food crops irrigated with waste water in Beijing, China.
Environmental pollution, 152 (3), 686-692. https://doi.org/10.1016/j.envpol.2007.06.056.
Khan, R. U., Nkousefat, Z., Tufarelli, V., Naz, S., Javdani, M. and Laudadio, V. (2012). Garlic (
Allium
sativum
) supplementation in poultry diets; effects on production and physiology. World’s
Poultry Science Journal, 68 (3), 417-424. https://doi.org/10.1017/S0043933912000530.
Nutrición Animal Tropical
_________________________________________________________________________________________________________________
________________________________________________________________________________________________
Nutrición Animal Tropical 19 (2): 63-84. ISSN: 2215-3527 / 2025
80
Kim, H. N., Lee, M. H., Kim, H. J., Kim, J. S. and Yon, J. (2008). A new trend in rhodamine-based
chemosensors: application of spirolactan ring-opening to sensing ions. Chemical Society
Reviews, 37 (8), 1465-1472. https://doi.org/10.1039/B802497A.
Mahrose, K. M., Abd-Elhack, M. E., Mahgoub, S. A. and Attia, F. A. (2019). Influence of stocking
and dietary probiotic supplementation on growing Japanese quail performance. Anais da
Academia Brasileira de Ciencias, 91, e20180616. https://doi.org/10.1590/0001-
3765201920180616.
Nandi, A., Yan, L. J., Jana, C. K. and Das, N. (2019). Role of catalase in oxidative stress-and-age-
associated degenerative diseases. Oxidative medicine and cellular longevity.
https://doi.org/ 10.1155/2019/9613090.
Nishikimi, M., Appaji, N. and Yagi, K. (1972). The occurrence of superoxide anion in the reaction
of reduced phenazine methosulfate and molecular oxygen. Biochemical and Biophysical
Research Communications, 46 (2), 849-854. https://doi.org/10.1016/S0006-
291X(72)80218-3
Olabode, A. D. and Adeleke, E. A. (2017). Rural climate and adaptive strategies for sustainable
food security in Akungba, Akoko, Nigeria. Analele Universităţii din Oradea, Seria
Geografie, 1, 7-16.
Oleforuh-Okoleh, V., Ndofor-Foleng, H. and Jude, T. (2015). Evaluation of growth performance,
haematological and serum biochemical response of broiler chicken to aqueous extract of
ginger and garlic. The Journal of Agricultural Science, 7 (4), 167-173. https://doi.org/
10.5539/jas.v7n4p167.
Omer, H. A., Ahmed, S. M., Abdel-Magid, S. S., El-Mallah, G. M., Bakr, A. A. and Abdel-Fattah, M.
M. (2019). Nutritional impact of inclusion of garlic (
Allium sativum
) and/or onion (
Allium
cepa
L.) powder in laying hens’ diets on their performance, egg quality and some blood
constituents. Bulletin of the National Research Centre, 43, 1-9.
https://doi.org/10.1186/s42269-019-0061-6.
Oluwafemi-Ayeni et al. Haemato-biochemical and antioxidant status in layers fed with garlic composite leaf mix.
_________________________________________________________________________________________________________________
________________________________________________________________________________________________
Nutrición Animal Tropical 19 (2): 63-84. ISSN: 2215-3527 / 2025
81
Piotrowska, A., Burlikowska, K. and Szymeczko, R. (2021). Changes in blood chemistry in broiler
chicken during the fattening period. Folia Biologica (Krakow), 59 (3-4), 183-187.
https://doi.org/ 10.3409/fb59_3-4.183-187.
Rehman, A., Setter, S. M. and Vue, M. H. (2011). Drug-induced glucose alterations part 2: drug-
induced hyperglycemia. Diabetes Spectrum, 24 (4): 234-238.
Roberts, D., Veeramachaneni, D. N. R., Schlaff, W. D. and Awoniyi, C. A. (2000). Effects of chronic
dietary exposture to genistein, a phytoestrogen during various stages of developments
on reproductive hormones and spermatogenesis in rat. Endocrine, 13, 281-286.
https://doi.org/10.1385/ENDO:13:3:281.
Sameer, A. S. (2013). Colorectal cancer: Molecular mutations and polymorphisms. Frontier in
oncology, 3, 114. https://doi.org/ 10.3389/fonc.2013.00114.
Schmidt, E. M. S., Paulillo, A. C., Vieira, G. R., Lapera, I. M., Pereira, A. J., Junior, L. N., Janine, D.
and Fagliari, J. J. (2009). Haematology of the bronze turkey (
Meleagris gallopavo
):
Variation with age and gender. International Journal of Poultry Science, 8 (8), 752-754.
http://dx.doi.org/ 10.3923/ijps.2009.752.754.
Thampi, N. and Jeyadoss, V. S. (2015). Comparative investigation of total antioxidant and free
radical scavenging activities of two allium species. The Asian Journal of Pharmaceutical
and Clinical Research, 8 (4), 148-151.
Weiss, C., Maker, H.S. and Lehrer, G.M. (1980). Sensitive fluorometric assays for glutathione
peroxidase and reductase. Analytical Biochemistry, 106 (2), 512-516.
https://doi.org/10.1016/0003-2697(80)90556-4.
Windisch, W., Schedle, K., Plitzner, C. and Kroismayr, A. (2008). Use of phytogenic products as
feed additives for swine and poultry. Journal of animal science, 86 (suppl-14), E140-E148.
https://doi.org/ 10.2527/jas.2007-0459.
Yoon, E., Babar, A., Choudhary, M., Kutner, M. and Pyrsopoulos, N. (2016). Acetaminophen-
induced hepatotoxicity: a comprehensive update. Journal of clinical and translational
hepatology, 4 (2), 131. https://doi.org/ 10.14218/JCTH.2015.00052.
Nutrición Animal Tropical
_________________________________________________________________________________________________________________
________________________________________________________________________________________________
Nutrición Animal Tropical 19 (2): 63-84. ISSN: 2215-3527 / 2025
82
Younus, H. (2018). Therapeutic potentials of superoxide dismutase. International Journal of Health
Sciences, 12 (3), 83.
