Nutritional Status and Reproductive Performance Evaluations in Birds Fed Diets Incorporated with Moringa oleifera Leaves and Seeds

Abstract

Introduction: This research assessed the impact of adding Moringa oleifera leaf and seed to the diet of laying hens on their nutritional status and reproductive performance. Methods: Twenty one (21) week-old laying hens were randomly divided into seven groups of three birds. The control group was fed Layers’ Mash, whereas the experimental groups were given a control diet supplemented with 5%, 10%, or 20% Moringa oleifera leaves and seeds. This feeding lasted six weeks. Eggs, meat and blood samples were analysed for nutritional quality evaluation and effect on some common reproductive hormones using appropriate standard protocols. Results: The present study revealed statistically significant (P < 0.05) differences in the internal and exterior properties of chicken eggs across groups. Compared with those in the control group, the concentrations of serum follicle stimulating hormone (FSH) and luteinising hormone were found to increase at some supplementation doses. Compared with those in the control group, significant (P < 0.05) increases in the serum total protein, albumin, ferritin, vitamins, and certain minerals were detected at different concentrations. Compared with those in the control group, significant (p < 0.05) changes in the dry matter content, energy content, ash content, ether extract content, albumen height of the bird meat and haematological parameters were detected. Conclusion: The addition of Moringa oleifera leaf and seed to the diet of laying birds increases their nutritional condition and reproductive performance, making it highly recommended.

Keywords

Nutritional status reproduction laying hen diet

Introduction

Achieving food security is crucial for any nation, which is why both developed and developing countries strive to enhance their food production capacities.^1,2 However, hunger and malnutrition persist in many developing nations, severely affecting low-income families.³ These issues have detrimental impacts on residents’ reproductive abilities and productivity, hindering overall economic progress. Poverty is closely linked to hunger and malnutrition, with unemployment and hyperinflation being the primary causes of hunger, while inadequate diets lead to malnutrition, resulting in long-term, fatal effects on individuals in many developing countries.^4,5

Medical and anthropometric evidence has demonstrated a strong correlation between malnutrition and infant mortality, stunted growth in children, and a weakened immune system in adults.⁶ Starvation undermines an economy, damages children's physical and mental development, and depletes society's future productive human capital.^7,8 Efforts to address nutritional security have been insufficient, as the focus has not adequately addressed the population's access to and quality of food.^9,10 Consequently, diets have become less diverse and nutrient-dense, negatively impacting on human health.⁴

Undernutrition and infection are known to affect both immunological and non-immunological defenses. This leads to increased frequency, intensity, and duration of common illnesses.¹¹ Vegetables and fruits, which are rich in phytochemicals, minerals, and vitamins, are vital for health. For example, rural inhabitants in developing countries obtain significant amounts of calcium and iron from vegetables.^12–14

Moringa oleifera (MO) is widely regarded as one of the most valuable medicinal plants due to its extensive therapeutic applications and exceptional nutritional profile.¹⁵ Every part of this versatile tree—leaves, seeds, pods, and bark—have been utilized for a wide range of domestic and medicinal purposes. The leaves and seeds, in particular, are rich in phytochemicals, essential vitamins, and minerals such as calcium, potassium, beta-carotene, and protein, which contribute to its nutritional importance.¹⁶ This nutrient-dense composition makes Moringa oleifera a valuable resource in addressing malnutrition, especially among vulnerable populations like infants and breastfeeding mothers.¹⁷

From a nutritional perspective, the bioactive compounds in Moringa oleifera, such as vitamins A, C, and E, play a critical role in neutralizing oxidative stress. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and the body's ability to detoxify these harmful by-products. Elevated levels of ROS can lead to cellular damage, contributing to the progression of chronic diseases, aging, and impaired reproductive function¹⁸ Vitamins A and E, present in high concentrations in Moringa oleifera, act as potent antioxidants positively modulating the activities of most enzyme antioxidants in the body.¹⁹ Vitamin A supports immune function and promotes healthy cell growth, while vitamin E inhibits the chain reactions initiated by free radicals, thereby preventing oxidative damage to cells and tissues.²⁰

In addition to vitamins, Moringa oleifera contains flavonoids and phenolic compounds that contribute significantly to its antioxidant properties. These phytochemicals help scavenge free radicals, reduce lipid peroxidation, and enhance the activity of endogenous antioxidant enzymes like superoxide dismutase and catalase, which further mitigate oxidative stress.¹⁶ This protective action is crucial in maintaining cellular integrity and promoting overall health, particularly in reproductive tissues where oxidative stress can impair fertility and reproductive outcomes.

Moreover, the presence of riboflavin (vitamin B2) in Moringa oleifera supports neuronal health and the development of a growing fetus, indicating its broader importance for both maternal and fetal well-being.²¹ Riboflavin plays a key role in cellular energy production and the reduction of oxidative stress by promoting the regeneration of glutathione, a vital intracellular antioxidant. The multi-dimensional anti-oxidant effect of Moringa oleifera extract goes down to regulation of gene expression.²²

Given its rich nutritional composition, Moringa oleifera has been linked to improved hormonal balance too. It influences the secretion of gonadotropins, which are critical for the development of gonads and the regulation of reproductive processes in mammals. By supporting the reproductive cycle and reducing oxidative damage to reproductive tissues, Moringa oleifera holds significant potential in enhancing fertility and overall reproductive health.²³ This study evaluated the effects of supplementing the diets of laying hens with Moringa oleifera leaves and seeds extracts on their nutritional status (egg and meat quality) and reproductive performance.

Materials and Methods

Collection of Plant Material

Moringa oleifera leaves and seeds were obtained from the Forestry Research Institute of Nigeria, Ahiaeke, Ndume, Umuahia, Abia State. The collected plant samples were identified by Dr Akinnibosun Henry Adewale of the Department of Plant Biology and Biotechnology, Herbarium Unit, Faculty of Life Sciences, University of Benin, Edo State, where a voucher specimen (UBH-M340) was deposited in the Department's herbarium.

The leaves were air-dried at room temperature, pulverized into a fine powder and stored in a dry, clean, sterile container for further use.

Plant Material

Leaves and seeds of Moringa oleifera were acquired from the Forestry Research Institute of Nigeria, which is located in Ahiaeke, Ndume, Umuahia, Abia State. Dr Akinnibosun Henry Adewale from the Department of Plant Biology and Biotechnology, Herbarium Unit, Faculty of Life Sciences at the University of Benin, Edo State, identified the plant samples. A voucher specimen (UBH-M340) was then placed in the Department's herbarium. The leaves were dehydrated by exposure to ambient air and then ground into a fine powder. The resulting powder was kept in a clean container free of moisture for future use.

Experimental Birds

The research used layers with a weight range of 250–350 g that were reared at the Poultry House of the Department of Animal Production and Livestock Management at the Michael Okpara University of Agriculture, Umudike. Prior to the experiment, the animals were acclimatised to their surroundings and nutrition for 7 days. They were kept in pens with metal demarcations in the animal house of the Department of Animal Production and Livestock Management at Michael Okpara University of Agriculture, Umudike, Abia State. During the entire period of the study, the test animals were provided with unrestricted access to both feed and tap water.

Experimental Design

Finely milled leaves and seeds were used in the formulation with 10% and 20% dietary incorporation of the growers’ mash. Commercially available standard growers feed (Vital Feeds) was purchased from accredited dealers The experimental animals were randomly divided into 7 groups of 3 animals in each group.

Group 1: Rat chow and tap water (normal control).

Group 2: 5% M. oleiferaleaf diet

Group 3: 5% M. oleifera seed diet

Group 4: 10% M. oleifera leaf diet

Group 5: 10% M. oleifera seed diet

Group 6: 20% M. oleiferaleaf diet

Group 7: 20% M. oleifera seed diet

Blood Collection

After the 28-day research period, blood samples were collected from the veins of the wing and placed in blood sample vials. The samples were then allowed to clot and centrifuged using a Uniscope Laboratory Centrifuge at a speed of 3000 rpm for 10 min. This process separated the plasma, which was kept at a temperature of −20 °C for biochemical analysis. The animals were eventually sacrificed by trained animal Scientists in the Department of Animal Science and Production, Michael Okpara University of Agriculture, Umudike. The birds were left without feed for 12 h but were allowed access to water. After which the weights were taken and sacrificed the following morning by cervical dislocation and quick cutting with very sharp in line with regulations according to American Veterinary Medical Association.²⁴ After feather-plucking, meat was were collected from breast muscle, thigh and other parts of the body and homogenized for proximate analysis

Biochemical Analyses

Serum Total Proteins and Albumin Concentrations

Serum total protein and albumin were assayed using spectrophotometric methods as described by Tietz et al,²⁵ as contained in the RANDOX test kits. Briefly, for total proteins, the following protocol applied. A volume (20 µl) of sample, standard and distilled water was dispensed into different tubes with micro-pipette. This was followed with the addition of 1000 µl of the reagent (Biuret reagent) mixed and incubated at room temperature for 30 min at 25 °C. At the end of the incubation period, absorbance of test and standard tubes were read at 520 s nm. The concentration was calculated from the standard.

For Albumins, the following protocol applied. A volume (3 µl) of sample, standard and distilled water was dispensed into different tubes with micro-pipette. This was followed with the addition of 1000 µl of the reagent (BCG concentrate) mixed and incubated at room temperature for 20 min. At the end of the incubation period, absorbance of test and standard tubes were read at 578 nm. The concentration was calculated from the standard.

Serum Ferritin Determination

Serum ferritin was determined according to the method described by White et al²⁶ as contained in Tulip Diagnostics kit. Briefly, the ferritin quantitative test kit is based on a solid phase enzyme linked immunosorbent assay. The assay system utilize some anti-ferritin antibody for solid phase (microtiter wells) immobilization and another mouse monoclonal anti-ferritin antibody in the antibody-enzyme (horse-radish peroxidase) conjugate solution. The test sample is allowed to react simultaneously with the antibodies, resulting in the ferritin molecules being sandwiched between the solid phase and enzyme linked antibodies. After a 60 min incubation at room temperature, the wells are washed to remove unbound labeled antibodies. A solution of TMB is added and incubated for 20 min, resulting in the development of a blue color. The color development is stopped with the addition of 2N HCL and the color is changed to yellow and measured at 450 nm using a microplater reader. The concentration of ferritin is directly proportional to the color intensity of the test sample.

Determination of FSH

This followed the method described by Uotila.²⁷ The FSH Quantitative Test Kit is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilize a polyclonal anti-FSH antibody or solid phase [microtiter wells]immobilization and a mouse monoclonal anti-FSH antibody in the antibody-enzyme [horseradish peroxides] conjugate solution.the test sample is allow to react simultaneously with the antibodies, resulting in FSH MOLECULES being sandwiched between the solid phase and enzyme-link antibodies. After 60 min incubation at room temperature, the wells are washed to remove umbound labeled antibodies. A SOLUTION of TMB is added and incubated for 20 min resulting in the development of a blue color the color development is stopped with the addition of 2N HCL and the color is change to yellow and measured spectorphotometrically at 450 nm. The concentration o FSH is directly proportional to the color intensity of the test sample.

Determination of LH

This followed the method described by Uotila.²⁷ Briefly, the essential reagent required for an immunoenzymometric assay include high affinity and specificity antibodies [enzyme and immobilized] with different and distinct epitope recognition, in excess, and native antigen, in this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated monocional anti-LH antibody.

Analysis of Micronutrient Content

The mineral analysis for inorganic phosphorus (P), magnesium (Mg), zinc (Zn), and iron (Fe) was conducted using an atomic absorption spectrophotometer (AAS) (Analytikjena AG, Germany).

Briefly, sample was made suitable for AAS analysis through the processes of filtration, digestion and dilution. Standard solutions of known concentrations of the metal(s) to be analyzed was prepared. Standards were used to calibrate the AAS instrument, creating a calibration curve by measuring the absorbance of each standard. The AAS instrument with the appropriate hollow cathode lamp for the metal of interest was set up. The correct wavelength for the metal being analyzed. This was followed by adjusting the flame type setting (air-acetylene in this case). The sample was introduced into the AAS instrument via the flame. The absorbance of the sample at the selected wavelength was measured. Concentration was determined from the calibration curve

The AOAC (2010) technique, was used to determine the concentration of vitamin A and E. In determination of vitamin A, the sample (0.1 ml) was measured into a test-tube I with a tight stopper and 1 ml of the KOH solution added. The tube was plugged and shaken vigorously for 1 min. The tube was put in a water bath at 60 °C for 20 min, then cooled. A quantity, 1 ml of xylene was added, plugged and shaken vigorously again for 1 min. The tube was centrifuged at1500×g for 10 min. The upper layer was collected into a glass and the absorbance A₁ measured at 335 nm against xylene. The sample was irradiated in the test tube II to the UV light for 30 min, and the absorbance A₂ measured. The concentration of vitamin A in the sample was calculated using the formula (A₁-A₂)×22.23.

Method for determination of vitamin E involves the conversion of ferric ions to ferrous ions by α-tocopherol and the formation of red colored complex with αα-dipyridyl. Absorbance of chromophore was measured at 520 nm in the spectrophotometer. Briefly, to 0.5 ml of serum, 1.5 ml of ethanol was added and mixed. To this was added 1.0 ml of αα-dipyridyl solution and 1.0 ml of ferric chloride solution and mixed. The color developed was read at 520 nm in the spectrophotometer. Values were read as mg/dl of serum from a standard curve.

Thiamine, riboflavin and pyridoxine were analyzed at the same time using High Performance Liquid Chromatography with UV detector (Agilent Technologies Model 1200, Germany).

Hematological Parameters

The complete blood count was assayed using an auto hematology analyser (Abbot Laboratories, Illinois). Briefly, labeled blood samples tube were placed in the designated sample loader of the analyzer. The analyzer aspirates a small volume of blood for analysis (20–50 µL). Blood sample is moved into different channels for measurement of various blood components. The analyzer processes the raw data to generate a detailed report of CBC parameters. Results are displayed on the analyzer screen, printed, or transferred electronically.

Statistical Analysis

Data obtained from the study were analyzed using IBM Statistical Product and Service Solution (IBM-SPSS) version 21 (Chicago, IL). Significant differences in the means were established by one-way analysis of variance (ANOVA), Post hoc multiple comparisons, and Duncan's homogenous subset. The results were expressed as means ± standard deviation of replicate measurements. Mean values with p ≤ 0.05 were considered significant.

Results

Table 1 below shows that Moringa oleifera leaf and seed incorporated diet improved the dry matter quality, ash/mineral, ether, and crude fiber contents of the groups incorporated different concentrations of the leaf and seed extracts compared to the control

Table 1.

Proximate Analysis of Moringa oleifera Leaves and Seeds Incorporated Diet.

	Dry matter	moisture	Ash/mineral	crude protein	ether extract	crude fiber	NFE	ME
Control	80.04 ± 0.20	8.96 ± 0.04	8.25 ± 0.02	21.88 ± 0.30	2.40 ± 0.02	4.15 ± 0.03	53.96 ± 0.1	3286.44 ± 0.40
5% Leaf	89.35 ± 0.20	10.65 ± 0.1	9.85 ± 0.04	18.38 ± 0.10	2.80 ± 0.04	5.00 ± 0.02	53.32 ± 0.1	3138.22 ± 0.20
5% seed	90.42 ± 0.40	9.58 ± 0.02	9.4 ± 0.06	20.13 ± 0.20	3.3 ± 0.01	4.45 ± 0.01	53.14 ± 0.2	3230.53 ± 0.20
10% Leaf	89.28 ± 0.10	10.72 ± 0.2	10.28 ± 0.10	17.50 ± 0.20	2.86 ± 0.01	5.65 ± 0.06	52.99 ± 0.2	3116.61 ± 0.10
10% Seed	90.21 ± 0.60	9.79 ± 0.04	9.62 ± 0.02	19.25 ± 0.40	3.42 ± 0.02	4.62 ± 0.04	53.30 ± 0.4	3212.72 ± 0.10
20% Leaf	89.15 ± 0.20	10.85 ± 0.2	10.64 ± 0.20	16.63 ± 0.20	2.89 ± 0.02	6.00 ± 0.02	52.99 ± 0.3	3093.17 ± 0.20
20% Seed	90.03 ± 0.40	9.96 ± 0.01	9.95 ± 0.04	18.33 ± 0.20	3.60 ± 0.06	4.85 ± 0.02	53.26 ± 0.3	3195.46 ± 0.20

Key: NFE: Nitrogen Free Extract, ME: Metabolizable Energy

Table 2 shows that Moringa oleifera leaf and seed incorporated diet improved the dry matter quality, ash/mineral, ether, NFE and ME of the bird meat in the groups fed different concentrations of the diet compared to the control

Table 2.

Proximate Analysis of the Meat of Birds Fed Moringa oleifera Leaves and Seeds.

	Dry matter	Moisture	Ash/Mineral	Crude protein	Ether extract	Crude fiber	NFE	ME
Control	76.24 ± 0.10	23.76 ± 0.10	0.84 ± 0.40	61.25 ± 0.20	6.24 ± 0.04	1.27 ± 0.03	6.65 ± 0.01	6.65 ± 0.01
5% MO leaf	77.42 ± 0.20	22.57 ± 0.40	1.14 ± 0.50	37.38 ± 0.20	7.00 ± 0.02	1.21 ± 0.02	30.69 ± 0.00	30.69 ± 0.20
5% MO Seed	78.32 ± 0.00	21.75 ± 0.10	1.15 ± 0.30	52.48 ± 0.10	10.02 ± 0.20	1.24 ± 0.02	13.43 ± 0.01	13.43 ± 0.10
10% MO leaf	77.83 ± 0.10	22.20 ± 0.10	1.64 ± 0.30	40.25 ± 0.40	11.50 ± 0.10	1.16 ± 0.00	23.28 ± 0.40	23.28 ± 0.40
10% MO seed	78.66 ± 0.10	21.34 ± 0.10	1.88 ± 0.10	53.38 ± 0.20	12.49 ± 0.20	1.20 ± 0.01	9.70 ± 0.01	3818.90 ± 3.00
20% MO Leaf	77.90 ± 0.20	22.10 ± 0.20	1.26 ± 0.20	37.63 ± 0.30	9.00 ± 0.02	1.18 ± 0.02	28.83 ± 0.20	3499.45 ± 7.40
20% MO Seed	79.38 ± 0.10	20.62 ± 0.10	1.50 ± 0.20	49.00 ± 0.10	10.51 ± 0.20	1.20 ± 0.02	17.19 ± 0.40	3725.07 ± 2.30

Key: NFE: Nitrogen Free Extract, ME: Metabolizable Energy

Table 3 shows the effect of MO leaves and the seed-supplemented diet on egg characteristics. Compared with those in the control group, the shell thickness, egg weight, egg length, egg volume, and shell weight in the MO-supplemented group and the MO-supplemented group (5% leaves) were significantly greater. Additionally, compared with those of the control group, the albumen weight and height, yolk weight, height and circumference, and Haugh unit significantly improved the tested parameters.

Table 3.

Egg Properties of Birds Fed Moringa oleifera Leaves and Seeds.

	Egg weight	Egg length	Egg volume	shell weight	S-thickness	Alb weight	yolk weight	Alb height	yolk height	yolk circum	Haugh unit
control	61.16 ± 0.2^d	56.45 ± 0.4^d	43.40 ± 0.4^c	7.59 ± 0.1^d	0.34 ± 0.0^c	39.63 ± 0.3^e	15.47 ± 0.4^d,e	10.63 ± 0.1^c	16.75 ± 0.4^c	39.93 ± 0.1^d	168.58 ± 0.2^c
5% MO Leaf	68.32 ± 0.4^a	58.83 ± 0.4^a	45.25 ± 0.2^a,b	8.68 ± 0.1^a	0.38 ± 0.0^a	42.48 ± 0.2^a	18.74 ± 0.1^a	11.68 ± 0.1^b	16.58 ± 0.1^c	41.80 ± 0.1^b,c	174.20 ± 0.4^b
5% MO Seed	66.65 ± 0.9^b	56.15 ± 0.9^d	45.30 ± 0.5^a	8.55 ± 0.0^a,b	0.35 ± 0.0^b,c	40.28 ± 0.2^d	17.13 ± 0.1^b	10.20 ± 0.1^d	18.53 ± 0.4^a	41.55 ± 0.6^c	167.93 ± 0.4^c
10% MO Leaf	63.92 ± 0.8^c	57.25 ± 0.5^c	43.45 ± 0.3^c	8.44 ± 0.1^b	0.34 ± 0.0^c	40.43 ± 0.4^d	16.59 ± 0.3^c	12.33 ± 0.4^a	16.05 ± 0.1^d	42.03 ± 0.2^a,b	175.62 ± 1.6^a
10% MO Seed	60.71 ± 0.3^d	55.40 ± 0.3^e	44.85 ± 0.1^a,b	8.15 ± 0.1^c	0.37 ± 0.0^a,b	37.13 ± 0.2^f	15.54 ± 0.1^d	12.15 ± 0.0^a	15.33 ± 0.1^e	42.23 ± 0.0^a	174.32 ± 0.1^b
20% Leaf	63.20 ± 0.4^c	57.45 ± 0.4^b,c	44.80 ± 0.2^b	6.76 ± 0.1^e	0.32 ± 0.0^d	40.77 ± 0.2^c	15.18 ± 0.0^e	11.83 ± 0.2^b	18.20 ± 0.2^a	38.08 ± 0.1^e	173.68 ± 0.7^b
20% Seed	63.91 ± 0.2^c	58.13 ± 0.2^a,b	45.00 ± 0.1^a,b	7.28 ± 0.1^f	0.34 ± 0.0^c	41.96 ± 0.1^b	14.61 ± 0.1^f	10.05 ± 0.0^d	17.65 ± 0.1^b	38.00 ± 0.1^e	166.88 ± 0.2^d

The values are the means ± standard deviations. The values in a column with the different superscripts are not significantly different (p > 0.05).

Key: alb-weight: albumen weight, alb-height: albumen height

Table 4 shows the effects of the incorporation of Moringa leaves and seeds on blood indices in birds. The results indicated significant (p˂0.05) increases in most haematological parameters in the test group compared to those in the normal control group.

Table 4.

Hematological Analysis of Birds Fed Moringa oleifera Leaves and Seeds.

	PCV	HB	RBC	WBC	MCV	MCH	MCHC	Lymphocytes	Eosinophils	Monocytes	Basophils	Heterophils
Control	27.33 ± 0.6^b,c	8.47 ± 0.3^c	2.92 ± 0.0^c	37.41 ± 2.2^c	93.71 ± 1.1^c	29.03 ± 0.9^b	30.98 ± 1.1^b,c	8.00 ± 7.0^a	26.67 ± 44.5^a	4.33 ± 5.8^a	31.33 ± 33.5^a	28.33 ± 39.9^a,b
5% Leaf	29.00 ± 1.0^a,b	9.03 ± 0.3^c	2.88 ± 0/0^c	40.67 ± 0.9^b	100.57 ± 3.0^a,b	31.33 ± 1.1^a	31.19 ± 1.9^b,c	30.00 ± 39.5^a	10.33 ± 8.0^a	34.00 ± 37.4^a	25.33 ± 42.1^a	4.00 ± 5.2^b
5% Seed	27.33 ± 0.6^b,c	8.47 ± 0.2^c	2.92 ± 0.0^c	34.17 ± 1.4^d	93.60 ± 1.2^c	29.00 ± 0.6^b	30.99 ± 0.8^b,c	3.67 ± 4.6^a	28.67 ± 35.0^a	25.00 ± 41.6^a	9.33 ± 7.4^a	32.33 ± 35.6^b,c
10% Leaf	29.00 ± 1.0^a,b	9.57 ± 0.3^b	2.99 ± 0.1^b	40.59 ± 2.4^b	97.04 ± 4.5^b,c	31.99 ± 0.3^a	33.02 ± 1.7^a,b	35.33 ± 37.2^a	4.33 ± 5.8^a	10.00 ± 8.5^a	27.33 ± 38.1^a	25.33 ± 42.1^a,b
10% Seed	30.33 ± 1.5^a	8.83 ± 0.2^c	2.88 ± 0.0^c	33.83 ± 1.3^d	105.34 ± 5.8^a	30.67 ± 0.5^a,b	29.18 ± 1.9^c	15.67 ± 3.1^a	11.00 ± 1.0^a	1.00 ± 0.0^a	1.33 ± 0.6^a	71.00 ± 4.6^a
20% Leaf	3.00 ± 1.0^a	10.20 ± 0.4^a	3.14 ± 0.0^a	47.21 ± 1.3^a	95.34 ± 3.2^b,c	32.42 ± 1.3^a	34.01 ± 0.9^a	21.00 ± 2.0^a	12.33 ± 1.5^a	1.33 ± 0.6^a	1.33 ± 0.6^a	64.00 ± 2.6^a
20% Seed	27.00 ± 0.00^c	8.50 ± 0.4^c	4.00 ± 0.0^c	33.67 ± 0.1^a	92.36 ± 0.5^c	29.08 ± 1.5^b	31.48 ± 1.6^b,c	15.67 ± 3.1^a	11.00 ± 1.0^a	1.00 ± 0.0^a	1.33 ± 0.6^a	74.00 ± 1.7^a

The values are the means ± standard deviations. The values in a column with the different superscripts are not significantly different (p > 0.05).

Compared with the control group, the 20% MO leaf- and seed-supplemented diet group showed significant (p < 0.05) increases in the levels of LH, followed by the 10% MO leaf- and seed-supplemented diet group (Figure 1). There was no significant difference in the concentrations of follicle-stimulating hormone across all dosages compared to those of the control.

Figure 1.

Serum concentrations of luteinising hormone and Follicule-stimulating hormone in birds fed solutions with varying doses of MO leaves and seeds.

Figure 2 below shows that the serum total protein, albumin (at 10% and 20%) and ferritin levels were significantly (p < 0.05) greater in all groups fed diets supplemented with varying doses of MO leaves and seeds than in the control group.

Figure 2.

Serum albumin, total protein and ferrittin concentrations of birds fed doses of MO leaves and seeds.

Table 5 shows serum concentrations of vitamins A, E and B1, B2 and B3 in birds fed different concentrations of MO in leaves and seeds. Result showed that the serum concentrations of vitamins A and E were significantly (p < 0.05) greater in birds fed MO at different concentrations in the leaves and seeds than in the normal control birds. Similarly, there was significant (p < 0.05) increase in the serum concentrations of vitamins B1 and B2 in most groups fed various doses of diets supplemented with MO leaves and seeds. The vitamin B6 concentration increased in birds fed 5% and 10% MO leaves compared to the control group.

Table 5.

Serum Concentrations of Vitamins B1, B2, B3, A and E in Birds Fed MO Seeds and Leaves.

Group	Vit B1 mg/dL	Vit B2 mg/dL	Vit B6 mg/dL	Vit A ug/dL	Vit E mg/dL
Control	1.05 ± 1.05^ab	0.997 ± 0.12^ab	0.246 ± 0.13^a	1.837 ± 0.32^a	0.027 ± 0.01^b
5% seed	0.98 ± 0.09^a	0.862 ± 0.42^a	1.026 ± 0.82^b	2.233 ± 0.25^a	0.027 ± 0.01^b
5% leaf	1.20 ± 0.20^b	1.252 ± 0.15^ab	0.333 ± 0.16^a	2.263 ± 0.20^a	0.010 ± 0.00^a
10% seed	1.06 ± 0.10^ab	1.145 ± 0.26^ab	0.771 ± 0.75^a	3.010 ± 0.26^b	0.047 ± 0.15^c
10% leaf	1.23 ± 0.07^bc	1.410 ± 0.41^bc	0.333 ± 0.28^b	2.997 ± 0.07^b	0.057 ± 0.01^c
20% seed	1.37 ± 0.06^c	1.859 ± 0.19^c	0.220 ± 0.19^a	2.437 ± 0.21^ab	0.060 ± 0.00^c
20% leaf	1.30 ± 0.10^c	1.109 ± 0.62^ab	0.289 ± 0.15^c	3.130 ± 0.72^c	0.060 ± 0.01^a

The values are the means ± standard deviations. The values in a column with the different superscripts are not significantly different (p > 0.05).

Table 6 shows the serum concentrations of Zn, Mg, iron and phosphorus in birds fed varying doses of MO in seeds and leaves. It shows that the serum concentrations of zinc and iron significantly (p < 0.05) increased in birds fed 5% and 10% MO, and the concentrations of magnesium and phosphorus in birds fed various concentrations of MO in seeds and leaves did not significantly differ from those in the control group.

Table 6.

Serum Concentrations of Zn, Mg, Iron and Phosphorus in Birds Fed Varying Doses of MO in Seeds and Leaves.

Group	Zn ug/dL	Mg mg/dL	Fe ug/dL	Inorganic phosphate mg/dL
Control	112.550 ± 3.74^b	1.933 ± 0.07^a	27.237 ± 1.27^ab	1.523 ± 0.06^c
5% seed	124.773 ± 8.77^bc	1.890 ± 0.14^a	27.030 ± 1.25^a	1.743 ± 0.15^c
5% leaf	125.220 ± 14.0^bc	1.830 ± 0.60^a	27.667 ± 1.95^b	1.553 ± 0.10^c
10% seed	184.997 ± 7.62^d	1.767 ± 0.51^a	22.903 ± 2.67^ab	0.907 ± 0.59^b
10% leaf	138.553 ± 24.64^c	1.733 ± 0.14^a	33.933 ± 2.96^c	0.500 ± 0.17^ab
20% seed	78.887 ± 6.71^a	2.387 ± 0.70^a	34.397 ± 3.51^c	0.427 ± 0.14^a
20% leaf	90.773 ± 6.18^a	1.983 ± 0.61^a	29.930 ± 2.80^bc	0.520 ± 0.13^ab

The values are the means ± standard deviations. The values in a column with the different superscripts are not significantly different (p > 0.05).

Discussion

In this study, the impact of a diet supplemented with Moringa oleifera (MO) seeds and leaves on the nutritional status and reproductive performance of laying hens fed modified diets was evaluated. Meat quality, defined by its properties and perceptions, is crucial since meat is a nutrient-rich diet with adequate moisture content, making it a potential medium for bacterial proliferation.

This risk is worsened by the use of low-quality feed-grade nutrient mixes.²⁶ Consumers want and are entitled to food items that are safer and have enhanced nutritional content. To do this, it is necessary for food scientists and animal breeders to devise tactics that modify the nutritional composition of meat products to enhance their quality and extend their shelf life.

The results of this study indicated that dietary Moringa oleiferaleaf (MOL) improved the dry matter quality in the groups fed 20% and 10% MO seeds compared to the control group. However, the crude protein content was lower in the groups fed 20% MO leaves and seeds, whereas the 5% and 10% MO seed diets had better crude protein contents. This reduction in protein content may be due to the toxicity of MO leaves and seeds, as reported by Monera et al.²⁷ However, MO has been found to be very safe for use at the used doses. The medicinal and otherwise benefits of MO by far outweighs whatever toxicity it might possess.

Vitamin E is a vital component for the growth and reproduction of animals. MO cannot be synthesized by the animals themselves and is totally reliant on their food for supply.²⁸ The results of this research demonstrated that the addition of MO leaves and seeds to the meals resulted in a statistically significant (p < 0.05) increase in the levels of vitamins A and E compared to those in the control group, as shown in Table 5. This discovery is consistent with the research conducted by Kasolo et al,²⁹ who identified MO as a repository of essential nutrients. The composition of tocopherols (α, β, γ, and δ) and tocotrienols (α, β, γ, and δ) in meat is affected by the specific tocol compounds found in the animal diet. Vitamin E is essential for the development and functioning of all animal species. Insufficient levels of vitamin E may result in several illnesses, including exudative diathesis, nutritional muscular dystrophy, encephalomalacia, stunted growth, and impaired reproductive performance in poultry.^30,31 Moringa oleifera (MO) includes potent antioxidant phytochemicals and necessary antioxidant micronutrients that have a good impact on reproductive performance.³² The present investigation demonstrated enhanced reproductive performance in avian species that were provided with a meal enriched with MO leaves and seeds. This improvement was shown by a statistically significant (p < 0.05) increase in luteinising hormone levels compared to those in the control group and in line with reports from other researchers.³³ This finding aligns with the established advantages of antioxidants in improving reproductive health.³⁴ Compared with those in the control group, the groups in which MO leaves and seeds were supplemented showed significant (p < 0.05) increases in the serum total protein (TP), albumin (at 10% and 20% inclusion), and ferritin levels (Figure 2). Compared with birds with higher TP values, birds with serum TP concentrations below 3.5 g/dL had a reduced likelihood of recovering from sickness.³⁵ The serum total protein concentrations found in this investigation were within the usual range for healthy birds, suggesting that the food proteins employed in this experiment were nutritionally adequate. In addition, MO seeds are rich in essential oils and minerals such as calcium (Ca), magnesium (Mg), selenium (Se), phosphorous (P), and zinc (Zn), as well as vitamins A, C, E, B1, B2, and B3. These components highlight the potential of MO to enhance egg quality and numerous other factors related to egg quality. A previous study corroborated these results, suggesting that the addition of MO may improve nutritional composition and reproductive well-being, hence increasing the overall health and productivity of animals.^31,36 The haematological parameters examined in diets supplemented with varying levels of Moringa oleifera leaves and seeds (Table 4) indicated significant (p˂0.05) changes in the levels of red blood cells (RBCs), white blood cells, PCV, and haemoglobin (Hb). Additionally, there were no significant (p > 0.05) changes in other blood parameters (lymphocyte (L), neutrophil (N), eosinophil (E), monocyte (M), or basophil (B)) as a result of the inclusion of Moringa oleifera leaves and seed meal in the poultry diet at the end of the experimental period. The data presented in Table 3 demonstrate the impact of M. oleifera leaves and seeds on the features of eggs. Adding 5% MO leaves to the diet resulted in a substantial increase in shell thickness, egg weight, egg length, egg volume, and shell weight compared to those of the control group. Furthermore, there was a notable increase in albumen weight and height, yolk weight, height and diameter, and Haugh units compared to those of the control group.³⁷ The result of this study showed better performance than in the study by Ebenebe et al³⁸ who reported that the addition of MO leaves did not have any impact on the egg shape index, which is closely related to egg shell strength and quality. Conversely, Makanjuola et al,³⁹ reported that the inclusion of M. oleifera seed meal in layer feed resulted in a significant increase in shell thickness compared to diets containing 10% Moringa oleifera seed meal (MOSM) and compared to the control diet. MO at both 5% and 10% had similar effects on increasing eggshell thickness.

Conclusion

We conclude that incorporating MO into bird feed will most likely improve the nutritional status and reproductive performance of laying hens, leading to improved egg production and meat quality and ultimately contributing to food and nutritional sufficiency.

Limitations to the Study

This study was limited by lack of grant funding for the project. For that reason the study could not be extended beyond the stated period of 6 weeks. Again it could not extend the investigation to the offspring to evaluate possible impacts on genetics

Footnotes

Acknowledgements

The authors are grateful to AJE Curie for English editing services.

Data Availability Statement

All the data generated and pertaining to the findings of this research are included in the manuscript.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Simeon Ikechukwu Egba

Statement of the Ethical Standard

The study material was obtained from the College of Natural Sciences Research Ethics Committee, Michael Okpara University of Agriculture, Umudike, with approval number MOUAU-CREC-BCM0062.

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