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DOI: https://doi.org/10.51244/IJRSI.2025.1210000304
An eight (8) weeks study was conducted to investigate the effect of varying inclusion levels of mushroom
(Pleurotus ostreatus) meal on the haematology and serum biochemistry of broiler chicks. One-hundred-and
twenty-Ross 308-day-old broiler chicks were randomly assigned to four dietary treatments in a completely
randomized design, with 30 birds per treatment, and three replicates having 10 birds per replicate. Mushroom
meal was included at varying levels (0, 1, 1.5, 2%) in the formulated starter and finisher diets fed to the birds.
The birds were fed a starter diet for the first 4 weeks and a finisher diet for the last 4 weeks, twice daily (8:00
am and 5:00 pm), with free access to water. Five hundred grams (500g) of each dried sample of Oyster mushroom
and experimental diets were taken to the laboratory for the determination of their proximate composition. At the
end of the experiment, ten (10mls) of blood was collected from three birds from each treatment groups at the
end of the feeding trial via venipuncture of pinea vein using a syringe into EDTA and plain containers and taken
to the biotechnology laboratory centre for determination of haematology and serum profile, respectively. The
data collected were subjected to analysis using SAS 2023, and means were compared at a 5% probability level
using Fisher's least Significant difference (FLSD). The crude protein, ether extract, crude fibre, ash, and
metabolizable energy contents were found to be 24.70, 1.41, 8.00, 7.50 % and 2454.21kcal/kg respectively. The
haemoglobin, lymphocytes, and mean corpuscular heamoglobin concentration were significantly (P< 0.05)
influenced by the experimental diet, with the birds in T2 recording the highest haemoglobin and mean
corpuscular haemoglobin concentration (15.70 and 38.12g/dl, respectively). The levels of urea and creatinine
was significantly (P<0.05) highest (14.26, 0.51 mg/dl, respectively) in birds fed 2% of mushroom meal. The
cholesterol and calcium levels were significantly reduced (P < 0.05) as the inclusion level of mushroom
increased, with the lowest levels (240.7, 1.16 mg/dl respectively) observed at the 2% inclusion level. The
haematological profile of the experimental birds showed that the animals were in good physiological condition
throughout the period of study. Mushroom meal can therefore be included in broiler diets at a 2% inclusion level
for optimal performance.
: Biochemistry, Broiler-chicks, Haematology, Mushroom meal, Serum.
In recent years, there has been a growing focus on developing sustainable, functional feed solution that can
enhance the output, health, and product quality of poultry production (Suberu et al., 2024). More recently, interest
has grown in novel feed resources such as black soldier fly (Hermetia illucens) larvae, mealworms (Tenebrio
molitor), plant leaf proteins, algae, insect-based products and mushroom meal due to their dual roles as nutrient
sources and functional additives. Edible mushrooms and their by-products (whole powder, stem residues, spent
substrate) have become promising feed additives because they are nutrient-dense and rich in bioactive
compounds, such as β-glucans, other polysaccharides, phenolic antioxidants, trace minerals, and when added to
poultry diets, these compounds have various physiological effects (Suberu et al., 2024; Bormon et al., 2024).
A growing body of research indicates that mushroom supplementation can positively modulate several
haematological and biochemical traits in poultry. Bormon et al. (2024); Suberu et al. (2024) reported increases
in haemoglobin and erythrocyte indices, reduced total cholesterol, and improved antioxidant status in broilers
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and layers fed dietary inclusion of Pleurotus ostreatus residues. These effects have been attributed to the
immunomodulatory and “trained immunity” actions of mushroom β-glucans and polysaccharides, which can
alter leukocyte proportions, functional responsiveness, antioxidant polyphenols and micronutrients that protect
cellular membranes (including erythrocytes and hepatocytes) from oxidative damage (Bar-Dagan et al., 2023;
Suberu et al., 2024). While weight gain and FCR appear to be major parameters for the assessment of the
usefulness of a feed ingredient, blood parameters further authenticate nutrient absorption and utilization by the
animal, so blood parameters also show the merits of a feed ingredient and at the same time showcase the
enhancement of health status of animals that consume the feed.
Blood is a medium through which nutrients are conveyed to various parts of the body system of an animal and a
readily available and fast means of assessing the health and nutritional status of an animal on feeding trial
(Oloche et al., 2018). According to Kumar et al. (2021) haematological indices (e.g., haemoglobin concentration,
packed cell volume, red blood cell counts, leucocyte differentials) and serum biochemical markers (e.g., total
protein, albumin, urea, creatinine, aminotransferases, cholesterol, triglycerides, and mineral levels) are sensitive
indicators of metabolic status, organ function, immune competence, and nutritional adequacy in broiler chickens.
Monitoring these parameters provides insight into whether a dietary intervention is physiologically beneficial,
neutral, or deleterious. Therefore, haematology and serum biochemistry constitute essential endpoints when
evaluating novel feed ingredients or functional supplements (Karageorgou et al., 2024). It is against this
background that this study was conducted to evaluate the haematology and serum biochemistry of broiler chicks
fed mushroom meal at various inclusion levels.
The study was conducted at the Poultry Section of the Animal Science Teaching and Research Farm, Faculty of
Agriculture, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria. Awka lies within the Guinea Savanna
zone (latitude 6.25°–6.28′ N and longitude 7.00°–7.08′ E) with an elevation of about 300 m above sea level,
annual rainfall of 1,000–1,500 mm, and a mean daily maximum temperature of 27 °C (Abajue and Ewuim,
2018).
Oyster mushroom (Pleurotus ostreatus) was cultured on sorghum grains enriched with rice bran and CaCO₃
sterilized at 121 °C for 30 minutes, and inoculated with pure mycelium obtained from a local farm. Fruiting
substrates were prepared with sawdust, rice bran, and CaCO₃, sterilized at 100 °C for 4 hours, inoculated with
mushroom spawn, and incubated under 80% relative humidity for 6 weeks and sprinkled with water daily as the
mushroom shoots out towards by the sixth week. Mature mushrooms were harvested, oven-dried at 80 °C for 3
h, milled, and stored for diet formulation.
A total of 120-day-old Ross 308 broiler chicks were purchased from Amo Hatchery, Ibadan. On arrival, the
chicks were weighed and randomly allotted to the four dietary treatments: T1 (0%), T2 (1.0%), T3 (1.5%), and
T4 (2.0%) inclusion levels of mushroom meal. Each treatment was replicated three times with 10 chicks per
replicate in a completely randomized design. Birds were brooded for two weeks and reared under standard
management with water and feed supplied ad libitum. The experimental diets were formulated to contain 23%
crude protein (starter) and 20% crude protein (finisher) with mushroom meal inclusions as specified (T1 (0%),
T2 (1.0%), T3 (1.5%), and T4 (2.0%), the trial lasted for 8 weeks. The ingredient and chemical composition of
experimental diet is presented in Table 1.
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Crude Fibre
3.60
2.60
2.90
2.60
3.55
4.50
3.95
4.50
Ether Extract
4.61
4.38
6.13
5.25
4.47
5.50
4.13
5.38
Moisture
7.75
9.75
11.25
12.50
9.75
8.25
13.25
9.00
Ash
2.75
2.00
2.75
2.47
2.60
2.88
2.80
2.75
ME(Kcal/kg)
2879.25
2864.48
2849.03
2977.85
2973.23
2965.50
2963.83
2945.88
About ten (10mls) of blood was collected at the end of the feeding trial via venipuncture of pinea vein of three
birds randomly selected from each of the treatment groups using a syringe into EDTA and plain containers and
taken to the Biotechnology Centre at Nnamdi Azikiwe University Awka, for determination of haematology and
serum profile respectively. Packed cell volume was determined using the standard technique described by (Coles,
1986), haemoglobin concentration was assayed colourimetrically using the cyanomethaemoglobin method
(Drabkin, 1945). Red blood cell and white blood cell was measured with the aid of Neubaur counter
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(haemocytometer) (Schalm,1976). Total protein determination was carried out by the biuret method of (Lubran,
1978), serum albumin concentration was determined using bromocresol green method (Doumas and Peters,
1997), determination of serum cholesterol concentration was done using the method as described by (Abel et al.,
1952) and serum creatinine concentration was determined by the modified Jaffe method (Blass et al., 1974).
Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin
Concentration (MCHC) were calculated from Hb, PCV and erythrocyte concentration of blood (RBC) (Jain,
1986). The indices were derived using the following formulae:
(𝑃𝐶𝑉 × 10)
𝑀𝐶𝑉 (𝑓𝐿) =
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (× 10⁶/µ𝐿
(𝐻𝑏 × 10)
𝑀𝐶𝐻 (𝑝𝑔) =
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (× 10⁶/µ𝐿)
𝑔 (𝐻𝐵 × 100)
𝑀𝐶𝐻𝐶 ( ) =
𝑑𝐿 𝑃𝐶𝑉
Proximate composition of mushroom meal and diets were analyzed according to AOAC (2012), and
metabolizable energy was calculated using the Pauzenga (1985) equation, and NFE by difference. Data collected
were subjected to analysis of variance (ANOVA) using SAS (2023), and treatment means were separated using
Fisher’s Least Significant Difference (LSD) test at a 5% level of significance.
The proximate composition of Mushroom meal is presented in Table 2.
Table 2 Proximate Composition of Mushroom Meal
Parameters (%)
Mushroom Meal
Moisture
18.25 ± 0.25
Crude Protein
24.70 ± 3.51
Ether Extract (Fat)
1.41 ± 0.90
Crude Fibre
8.00 ± 0.60
Ash
7.50 ± 0.00
Nitrogen-Free Extract (Carbohydrate)
40.14 ± 3.21
*Metabolisable Energy (kcal/kg)
2454.21 ± 2.69
*ME = (37 × Crude Protein) + (81.8 × Crude fat) + (35.5 × NFE)
The crude protein level (24.70 %) confirms that mushroom meal is a valuable alternative protein source.
Comparable protein contents have been documented for Pleurotus ostreatus (21–28 %), Agaricus bisporus (20–
27 %), and Lentinus edodes (22–25 %) (Suberu et al., 2024; Bormon et al., 2024). These variation in protein
values among mushroom species and substrates reflects differences in cultivation media, harvest age, and drying
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methods (Rathore et al., 2017). Adebayo et al. (2021) in their study poibted out that mushroom protein is highly
digestible and rich in essential amino acids such as lysine, methionine, threonine, and valine, which are often
deficient in cereal-based broiler diets, and therefore its inclusion in poultry feed can help balance amino-acid
profiles and reduce reliance on costly soybean meal.
The ether extract value (1.41 %) indicates a low lipid content, consistent with the lean nature of mushrooms.
Most edible mushrooms contain 1–3 % crude fat, which is composed largely of unsaturated fatty acids such as
linoleic and oleic acids (Barros et al., 2008). Low lipid levels are advantageous for feed formulation because
they reduce oxidation risks and improve product stability. Nevertheless, Yildiz et al. (2017) documented that the
presence of essential fatty acids in mushroom lipids contributes to antioxidant potential and enhances flavour
acceptability in feed.
The crude fibre (8.00 %) observed is relatively high compared with conventional plant proteins but within the
range (6-10%) reported by Suberu et al. (2024) for mushroom by-products. Mushroom cell walls are rich in
chitin, β-glucans, and other non-starch polysaccharides that contribute to fibre content and functional benefits as
dietary β-glucans can act as prebiotics, improving gut health and modulating the immune response in poultry
(Bar-Dagan et al., 2023). However, Mthana and Mthiyane, (2024) pointed out that excessive fibre levels may
reduce nutrient digestibility if inclusion levels are not well balanced. Therefore, while mushroom meal provides
functional fibre, its dietary proportion must be optimized to avoid compromising energy utilization.
The ash content (7.50 %) indicates considerable mineral density. Mushrooms are recognised for their high
mineral content particularly potassium, phosphorus, magnesium, calcium, iron, and zinc (Rathore et al., 2017).
The ash content in the current study is within the range of 6-10% reported by Suberu et al. (2024), and Adebayo
et al. (2021) for Pleurotus and Agaricus species. Such mineral richness enhances the feed’s potential to support
bone development and metabolic processes in poultry.
The NFE value (40.14 %) reflects the carbohydrate fraction, mainly comprising glycogen-like polysaccharides,
hemicellulose, and soluble sugars. These carbohydrates provide an accessible energy source and may also
include prebiotic polysaccharides that promote beneficial gut microflora (Adebayo et al., 2021; Suberu et al.,
2024). The calculated metabolisable energy (2454 kcal/kg) lies within the range reported for mushroom meals
(2300–2600 kcal/kg) and is adequate to support moderate inclusion in broiler diets without adversely affecting
energy balance (Bormon et al., 2024). Although mushrooms are less energy-dense than cereal grains, their
functional components justify partial substitution for conventional energy and protein ingredients.
Table 3 shows the haematological parameters of broiler chicks fed varying levels of mushroom meal.
Table 3 Haematological Parameters of Broilers fed Varying Levels of Mushroom Meal
Parameter
T1
T2
T3
T4
SEM
LOS
Ref Value
PCV (%)
41.00
42.33
37.33
41.67
7.98
NS
25.20 - 45.20
Hb (g/dl)
15.30a
15.70a
14.20b
15.50a
0.66
*
10.02 - 15.1
RBC (1012/L)
4.17
4.40
4.13
4.33
0.30
NS
2.0 – 3.0
TWBC (109/L)
91.50
90.57
89.63
89.33
1.22
NS
20.0– 30.0
Lymphocytes (%)
61.20b
60.47b
63.97a
59.03b
1.17
*
50.0 - 75.0
Platelet (109/L)
40.30
40.00
39.33
39.23
1.01
NS
20.0– 30.0
MPV (fL)
13.07
13.07
12.87
12.90
0.19
NS
10.0 – 20.0
MCV (fL)
99.28
102.88
87.82
96.89
10.75
NS
90.0-130.0
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MCH (pg)
37.05
38.12
33.40
35.96
3.66
NS
28.0 - 34.0
MCHC (g/dl)
37.33b
38.12a
38.04a
35.96c
0.36
*
26.0 – 35.0
Granulocytes (%)
14.33
14.67
14.33
14.37
0.64
NS
20.0 – 40.0
abc Mean values with different superscripts within a row differed significantly; PCV= Packed Cell Volume;
Hb= Haemoglobin; RBC= Red Blood Cell; TWBC= Total White Blood Cell; MCV= Mean Corpuscular
Volume; MCH= Mean Corpuscular Hemoglobin; MCHC= Mean Corpuscular Hemoglobin Concentration;
SEM= Standard Error of Mean; LOS= Level of Significance; * Significant at 0.05; NS= Not Significant.
Feeding varying levels of mushroom meal to broiler chicks significantly (P< 0.05) influenced the Hb,
Lymphocytes and MCHC of broiler chickens. The other measured parameter numerically varied among the
treatment groups, but no significant differences were observed. The values of PCV, lymphocytes, MPV, and
MCV recorded by all the groups were, however, within the normal range for healthy chicken (Aeangwanich et
al., 2004; Ahmed, 2018), while the other parameters were outside the reference range reported by Aiello and
Mays (1998), and Okeudo et al. (2003) for broiler chicken.
Haematology is available in determining the physiological responses of chickens as the blood constituents are
the biochemical medium of transportation in all animals, and thus their profile shows the health status of the
birds (Akintomide et al., 2021; Oyebode, 2015). The two significant parameters used in assessing the health
status of broiler chickens are the PCV and Hb. Packed Cell Volume (PCV) is involved in the transport of oxygen
and absorbed nutrients round the body, delivering it to target cells or tissues (Onunkwo et al., 2022). The PCV
value ranged between 37.33 - 42.33% and were within the normal range described by Ahmed, (2018) for a
healthy chicken. Feeding varied levels of mushroom recorded no significant effect on the PCV values of broiler
chickens in the present study.
Haemoglobin is the oxygen carrying protein in the RBC. Hb levels is a direct reflection of the amount of oxygen
in the blood. The Hb values were significantly (P <0.05) affected by levels of mushroom meal inclusion and its
values were slightly above the normal range described by Aeangwanich et al. (2004) and Ahmed, (2018), for
healthy birds. This elevated haemoglobin concentration suggests an improvement in oxygen-carrying capacity
and general erythropoietic efficiency. Similar findings were reported by Bormon et al. (2024), who observed
enhanced haemoglobin levels in broilers supplemented with Pleurotus ostreatus stem residue. The authors
attributed this to improved mineral bioavailability, particularly iron, and reduced oxidative degradation of red
blood cells due to antioxidant compounds in mushrooms. Suberu et al. (2024) also confirmed that mushroom
polysaccharides, phenolics, and vitamins protect erythrocyte membranes from oxidative stress, thereby
sustaining haemoglobin integrity.
The Hb values in these chickens correspond with their PCV values as chickens with higher PCV had higher
Haemoglobin concentrations (Jubril et al., 2022). These findings are in line with the report by Kumar et al.
(2021), who also observed a corresponding higher Haemoglobin concentration in broiler birds with higher PCV.
Lakurbe et al. (2018) documented that the range of values obtained in the present study could indicate that the
birds were adequately nourished and thus not anaemic or showing any sign of disease infection or parasite
infestation.
The lymphocytes were significantly (P<0.05) influenced by varied levels of mushroom meal and the values were
within the normal range of 50.0-75.0 documented by Ahmed, (2018) for a healthy chicken. According to Guyton
and Hall (2006), an increase in lymphocytes in the blood indicates enhanced immunological well-being of the
body.
The MPV and MCV were not influenced by mushroom meal, but their values were within the normal range
reported for a healthy chicken. MCH indicates the blood-carrying ability of RBC. The study reveals that the
mushroom increased the blood-carrying ability of the RBC, with the highest value recorded, which is slightly
above the reported normal range for a healthy chicken (Ahmed, 2018).
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The serum metabolites of broiler chicks fed varying levels of mushroom meal is shown in Table 4.
Table 4. Serum Metabolites of Broilers fed Varying Levels of Mushroom Meal
Parameter
T1
T2
T3
T4
SEM
LOS
Ref Value
Total Protein (g/dl)
3.10
3.15
3.16
3.24
0.32
NS
3.0 -5.5
Globulin (g/dl)
1.83
2.14
1.92
2.20
0.25
NS
1.5 – 3.5
Albumin (g/dl)
1.27
1.01
1.24
1.04
0.16
NS
1.0 – 3.0
Urea (mg/dl)
13.06b
12.04b
12.67b
14.26a
0.60
*
2.0 – 8.0
Creatinine (mg/dl)
0.35b
0.45a
0.46a
0.51a
0.07
*
0.2 – 0.5
ALT (U/L)
3.33
4.67
3.00
4.33
1.42
NS
5.0 – 20.0
AST (U/L)
110.67
112.83
119.67
122.17
9.61
NS
100 – 300
Triglycerides (mg/dl)
191.56
193.56
191.11
178.22
9.31
NS
30 -150
Cholesterol (mg/dl)
455.3a
440.7a
309.3a
240.7b
80.16
*
100 – 200
Calcium (mg/dl)
8.48a
6.15b
1.99c
1.16c
0.54
*
8.0 – 12.0
abc Mean values with different superscripts within a row differed significantly; AST = Aspartate Amino
Transferase; ALT = Alanine Amino Transferase; SEM= Standard Error of Mean; LOS= Level of Significance;
* Significant at 0.05; NS= Not Significant.
The results of the serum metabolite analysis show that most of the parameters tested in this study were not
influenced by dietary treatments; only a significant (P<0.05) increase in the level of urea, creatinine and a
reduction of cholesterol and calcium concentration in blood serum was observed in response to inclusion of
mushroom in the diet. The levels of total protein, Albumin, and globulin in this study fell within the normal range
reported by Mitruka and Rawnsley, (1977).
The use of biochemical indices as a pointer to conditions that may not be readily noticed by performance indices
cannot be overemphasised (Oloche et al., 2018). According to Upah et al. (2024), the plane of nutrition is known
to affect these values. Total protein, globulin, and albumin are critical for maintaining physiological functions
and immunity, and their levels in this study did not show significant differences across the treatments (T1-T4),
but their values are within the reference ranges reported by Mitruka and Rawnsley, (1977) for a healthy chicken.
Similar findings have been reported by Suberu et al. (2024), who found that dietary supplements, including
mushroom extracts, did not significantly alter serum protein levels in broilers. This stability suggests that
mushroom meal, within the levels tested, does not adversely affect protein synthesis or liver function. The total
protein (TP) content numerically increased in the treated group of broiler chicken as the inclusion level increased
in this study. The reason for the higher plasma protein accretion in blood vessels might be a result of mushroom
supplementation in broiler diets. Shang et al. (2014) reported that mushrooms can increase the availability of
amino acids (methionine and cysteine) for protein formation in broiler chickens.
Significant (P<0.05) differences were observed in urea and creatinine levels. The urea level was significantly
(P<0.05) higher in T4 compared to other treatments, while the inclusion of mushroom meal significantly
(P<0.05) increased creatinine levels in the birds than those on the control diet. Their levels exceeded the reference
ranges reported by Meluzzi et al. (1992) for a healthy chicken. Elevated urea and creatinine levels can indicate
dehydration, renal stress or impaired kidney function. Research by Olanrewaju et al. (2022) suggests that
highprotein diets can increase urea and creatinine levels due to higher nitrogenous waste production. The increase
in urea and creatinine in the treatment groups might be a result of higher levels of mushroom meal.
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Alterations in the activities of serum enzymes, such as Aspartate aminotransferase (AST), Alanine
aminotransferase (ALT), are often utilized as markers of toxicity for the assessment of biochemical and
physiological health of vital organs and tissues, such as liver and heart (Akinsulie et al., 2021). Upah et al. (2024)
established that damage to the cellular membrane in tissues where these enzymes are normally localized results
in their leakage into the bloodstream, giving an index of cellular integrity. The results of this study showed that
mushroom meals had no significant (P > 0.05) effect on the levels of AST and ALT, but the ALT value falls
within the normal range documented by (Meluzzi et al., 1992), while the AST levels fall below the normal range
reported by (Meluzzi et al., 1992). The values of the liver enzymes in this study showed that they were not
influenced by the anti-inflammatory activities of the phytochemicals, flavonoids and terpenes present in
mushroom meal.
Cholesterol levels were significantly (P<0.05) lower in T4 compared to the other treatment groups, and their
levels in the present study was higher than the range reported by Abdi-Hachesco et al. (2011) for the broiler
strain. Calcium levels were significantly (P<0.05) lower in T3 and T4 compared to T1 and T2, with T4 showing
the lowest levels. Lower cholesterol levels in broilers fed higher mushroom meal levels could be related to the
high fiber content or specific bioactive compounds found in mushrooms that affect lipid metabolism. This aligns
with findings by Duda et al. (2025), who reported that mushroom supplementation can lower serum cholesterol
levels due to their bioactive components. The drop in calcium levels in higher mushroom meal groups might be
attributed to the high phosphorus content of mushroom meal, which, according to the study of Mahfuz et al.
(2019), can affect calcium absorption.
From the results obtained, it can be concluded that the inclusion of mushroom meal at 2% in broiler diet resulted
in optimum performance of broiler chicks without any negative effect on the health status of the chicks.
The authors acknowledge the academic staff of the Department of Animal Science, Nnamdi Azikiwe University,
Awka, for providing constructive criticism, which improved the quality of the study.
This study adhered to ethical research standards as approved by the Ethical Committee of the Nnamdi Azikiwe
University, Nigeria, ensuring responsible data use, informed community participation, non-invasive
environmental practices, transparency and adhering to the Animal handling protocols.
The authors declare that this study has received no financial support.
The authors have no conflicts of interest to declare.
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