Response of Broiler Chickens to Oral Administration of Ocimum Gratissimum Leaf Extracts

The study was carried out on one hundred and ninety-ve day-old broiler chicks of cobb500 to study the effect of Ocimum gratissimum leaf extracts on growth performance, blood prole, microbial population in the faecal and gut samples. The chicks were randomly selected into ve groups with thirty-nine chicks per group and each group replicated thrice. The groups were: control (synthetic antibiotics), extracts from 200 g fresh leaf, 400 g fresh leaf, 40 g air-dried leaf and 80 g aid-dried leaf per litre of water respectively. Data collected were subjected to one-way Analysis of Variance. The growth performance except mortality was similar (P>0.05) across the groups. Albumin, urea, cholesterol, alkaline phosphate and sodium were inuenced (P<0.05) at the starter phase while only alkaline phosphate was signicantly (P<0.05) highest in birds on 400 g of fresh leaf extract at the nisher phase. Neutrophil was highest (P<0.05) in birds on 400 g of fresh leaf extract while those on 200 g had higher values of lymphocytes and eosinophil at starter phase. At nisher phase, birds on antibiotics and 80 g of air-dried leaf extract had higher (P<0.05) white blood cell. Faecal total microbial population was least (<0.05) in birds on antibiotics and 200 g fresh Ocimum gratissimum leaf extract at starter phase. The study concluded that the adoption of Ocimum gratissimum leaf extract as prophylactic treatment against bacteria should be encouraged among poultry farmers.


Introduction
Over the past few decades, antibacterial resistance has been a signi cantly concerning global health threat because bactericidal action of almost all known antibiotics is on the wane against pathogenic microorganisms in animal production (Reig and Toldra, 2008). Application of antimicrobial agents in poultry has been associated with the growth of resistant bacteria species. Resistant strains among poultry pathogens can lead to suppressed potency of antibiotics, translating to economic losses as resistant bacteria species pose signi cant risk to human health. Poultry is often raised under intensive conditions, primarily reared on large amounts of antimicrobials that are supplied to prevent, curtail or manage outbreak. (Marshall and Levy, 2011;Nhung, 2017).
Observed trend of research on application of antibiotic alternatives and feed additives to promote livestock growth have been explored with enhancement of gut health using low dosage of antibiotics.
Classes of antibiotic alternatives available to boost productivity aid maximization of genetic potential under existing commercial conditions in poultry, such as enzymes, antimicrobial peptides, organic acids, phytogenics, prebiotics, probiotics, symbiotics, hyperimmune egg antibodies, bacteriophages, clay and metals (Gadde et al., 2017). Natural herbal product has been explored in pursuit of improved health status among birds coupled with consumer satisfaction that is increasingly associated with healthier product (Gardzielewska et al., 2003). An evaluation of performance of broiler chickens reveal novel herbal supplementations are incorporated to boost indices among experimental chickens since blood contribute a signi cant role in health pro le assessment. Scent leaf (Ocimum gratissimum) possesses bioactive principles potent against diverse bacterial species (Iwalokun, 2003;Akinyemi et al., 2004). The leaves, owers, roots, and oils demonstrated antiviral, larvicidal, bactericidal, and insect repellant properties (Oparaocha et al., 2010;Sumitha and Thoppil, 2016;Pandey et al., 2017). The study of Junaid et al. (2006) showed that antimicrobial e cacy of cold-water extract of the fresh leaf of O. gratissimum applied on all test isolates had highest inhibitory activity. Mahomoodally (2013) likewise declared that the trend of application of medicinal substances points towards validating the e cacy and safety of African traditional medicines in relation to its form of application, signifying that the quality of principles utilized is dependent on the form of Ocimum gratissimum. On that premise, this study is designed to investigate the administration of raw and air-dried Ocimum gratissimum leaf extract on performance and blood pro le of broiler chickens.

Material And Methods
Experimental Site: The experiment was carried out at the Poultry unit of the Teaching and Research Unit of Directorate of University farms, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria. The site is located in the rain forest vegetation zone of south -western Nigeria on Latitude 7 0 10' N and Longitude 3 0 2'E and altitude of 76m above the sea level (Google Earth, 2020). The climate is humid, with a mean annual rainfall of about 1037mm and mean temperature and humidity of 34.7 0 C and 83%, respectively.
Preparation of the extracts: the extraction methods were two: the fresh and air-dried leave extracts. The fresh leaf extraction was prepared as follow; 200 and 400 g of fresh leaves was blended with six litre of water respectively. The procedure for the extraction of air-dried leaf extract is as follows; fresh leaves of the plant were harvested and spread in a room for air-drying. Air-drying method was adopted to prevent loss of volatile oils when spread under direct sunlight. The air-dried leaves were milled and dissolved in water at 40 and 80g (an equivalent of 200 and 400g fresh leaves, respectively) per six litres of water respectively. The solutions were stirred every 30 minutes for 3 hours and allowed to stand 24 hours. After the 24 hours, the solution was sieved to get extract according to the procedure of Jesuwenu and Okozi (2017).

Management of Experimental Birds
A total of one hundred and ninety-ve (195) day old chicks of Cobb 500 strain of broiler chickens was purchase from a reputable hatchery. The birds were randomly divided into ve experimental groups. Each group was further sub-divided to three replicates of thirteen chicks per each. The groups were: control (use of synthetic antibiotics), extracts from 200 g fresh leaf, 400 g of fresh leaf, 40 g of air-dried leaf and 80 g of aid-dried leaf respectively. The extract was served to the birds at the rate of one-third of daily water intake throughout the experimental period expect a day prior and the day of vaccination. Water was served immediately the birds in each replicate nished the served extract. Brooding of chicks lasted for two weeks in individual pen with the use of charcoal pots and electrical bulbs as heat source and lightings. Synthetic antibiotic (enro oxacin) was used for the birds in the control group. Commercial broiler starter diet was supplied for the rst four weeks of the study while commercial broiler nisher diet was used for the last three week. Feed and water were supplied ad-libitum. The experiment lasted for seven weeks.
Data collection: Growth performance indices (feed intake, weight gain) were taken weekly while feed conversion ratio was calculated by dividing feed intake by weight gain. Blood samples were collected on the 28 th (end of starter phase) and 49 th ( nisher phase) days of the experiment from each replicate.
Blood sample of 2.0ml was drawn via wing vein puncture with hypodermic syringes into bottles containing an anticoagulant; Ethylene diamine tetra acetate (EDTA) for the determination of haematological parameters. Packed cell volume (PCV), haemoglobin (Hb) concentration, red blood cells (RBC) and white blood cells (WBC) and differential counts were determined according to the procedure of Schalm et al. (1975); mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were derived by calculation from the RBC, Hb, PCV values as described by Jain (1986). Another 2.0ml of blood was drawn into labelled sample bottles without EDTA for serum biochemical indices determination. Serum was separated from the clot blood by centrifuge. Serum indices; Total protein (TP) and albumin values were determined by Biuret method. Globulin was calculated as the difference between total protein and albumin. Collection of faecal and gut samples: On the 28 th day of the experiment, black nylon was spread on the litter in each pen. Each pen was observed and the birds were allowed to roam freely over the nylon. The nylon was then withdrawn from the pen as soon as the feacal sample were collected on the nylon. Each sample collected was kept in a universal bottle. On the 49 th day of the experiment a chicken of average weight of each replicate was picked, fasted overnight and slaughtered. The gut sample was aseptically collected from the slaughtered bird for all replicates. All samples were taken to the laboratory for analysis to determine bacterial identi cation and count following procedure outlined by Adhikari and Kwon, (2017).

Determination of phytochemicals, microbial count and identi cation
Gram-positive and negative bacteria species were examined as caecal contents were serially diluted and plated, then incubated at 37°C under a microaerophilic condition for 15-18 hrs. Diluted samples were cultured on nutrient agar. Subsequently, adapted Mueller Hinton Agar (MHA) was modi ed according to Al-blooshi et al. (2021) to develop MHA-C15 (MHA containing 15% volume/volume water extract of clove). Gram-negative bacterial pathogens such as Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa grew on MHA-C15. However, none of the major Gram-positive bacterial pathogens such as Streptococcus faecalis and Clostridium perfrigens grew on it. To isolate bacteria in caecum-ilium mucosa, as well as the faeces, samples were rinsed in sterile Phosphate Buffered Saline (PBS) of pH 7.4 after luminal contents were separated three times, and homogenized in 20 ml of PBS. Samples extracted were kept in cold storage and transported to the laboratory prior to subsequent analysis. Thereafter, Isolation and identi cation was carried out by observing colony morphology, grams staining and standard cultural and biochemical test.
Statistical Analysis: Data collected were subjected to One-way Analysis of variance using SAS (2010). The signi cant means were separated at p<0.05 using Duncan's Multiple Range Test in the software package.

Result
Activity of Ocimum gratissimum leaf extract on growth performance of broiler chickens is documented in Table 1. All growth performance parameters examined ( nal weight, weight gain, feed intake feed conversion ratio and water intake) were not affected (p>0.05) by extract offered. However, mortality was signi cantly (P<0.05) highest among birds on 400 g of fresh leaf extract while the least was obtained from those on 80 g of air-dried leaf extract.  Among haematological parameters obtained, blood neutrophil, lymphocytes and eosinophil were in uenced (P<0.05) by the oral administration of Ocimum gratissimum leaf extract (Table 3). It was observed that neutrophil was higher in birds on 400 g fresh Ocimum gratissimum leaf extract but the least value was from the counterpart on 200 g fresh Ocimum gratissimum leaf extract. Lymphocytes and eosinophil were higher in bird on 200 g fresh Ocimum gratissimum leaf extract while the least value of the two measurements were obtained from birds on 400 g fresh Ocimum gratissimum leaf extract and 80 g air-dried Ocimum gratissimum leaf extract. The faecal microbial population of broiler chickens is presented in Table 4. The total bacterial count and clostridium spp and proteus were signi cantly (P<0.05) affected extract offered. Total microbial population was highest in birds on 40 g of air-dried Ocimum gratissimum leaf extract while the birds on antibiotics and 200 g fresh Ocimum gratissimum leaf extract had least value. There was no presence of Clostridium Spp in birds on antibiotics. Proteus mirabilis was also not present in the faecal samples of birds on 200 g fresh and 80 g air-dried Ocimum gratissimum leaf extract. The effect of oral administration of Ocimum gratissimum leaf extract on the serum biochemical parameters at nisher phase (Table 5) indicated that ALP was the only measurement that was in uenced (P<0.05). The highest value was recorded from chickens on 400 g fresh Ocimum gratissimum leaf extract while the least value was obtained from their counterpart on antibiotics. The effect of oral administration of Ocimum gratissimum leaf extract on haematological parameters of broiler chickens at the nishing phase is presented in Table 6. White blood cell count was the only parameter signi cantly (P<0.05) different across the treatment groups. Chickens on antibiotics and those on 80 g air-dried Ocimum gratissimum leaf extract had increased count than groups given 40 g air-dried Ocimum gratissimum leaf extract. Gut microbial count of broiler chickens offered Ocimum gratissimum leaf extract at nisher phase presented in Table 7 reveal extract did not affect bacterial species (E. coli, Streptococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Clostridium perfrigens and Proteus mirabis) and the subsequent total count.

Discussion
The similarities in the growth performance indices across the groups is an indication that the O. gratissimum leaf extract has antibacterial properties and this resulted into similar nal live weight. The oral administration air-dried leaf extract further established that the processing method (air-drying) might have reduced the toxicity of phytochemicals and therefore reduced the mortality percentage. Various antinutritional factors are present in plants reduce the digestibility of proteins and mineral absorption. Anti-nutrients are one of the key factors that reduce the bioavailability of various components in diets (Samtiya et al., 2020). Njoku et al. (2017) reported that air-drying method of O. gratissimum leaf is the best processing method to preserve yield and chemical composition of the essential oil for the plant to retain the components responsible for its biological activity. Among bacteria species examined, clostridium spp growth was inhibited by antibiotics when compared with count from birds offered extracts of Ocimum gratissimum. For proteus mirabilis, 200 g and 80 g of fresh and air-dried Ocimum gratissimum extracts respectively hampered the growth of Proteus mirabilis. Eugenol, according to Nakamura et al. (1999) suppress Proteus mirabilis growth. Hence, both level of extract su ciently hinder occurrence of Proteus sp. At the nisher phase, serum ALP followed the trend of the starter phase as birds on antibiotics, having suppressed ALP value at nisher phase. Reason proposed at the starter phase consequently extends to the nisher phase. Total bacteria count was lowered when birds were offered antibiotic and 200 g of air-dried Ocimum gratissimum extract -a consequence of the broad acting capacity of both substances. At the nisher phase, higher WBC among groups given antibiotics, 400 g of fresh Ocimum gratissimum extract and 80 g of air-dried Ocimum gratissimum extract than the 9.33 x 10 9 /L declared by Ajayi and Imouokhome (2015) was documented. WBC among chickens given 40 g air-dried Ocimum gratissimum extract was likewise lower than the 6.87 -9.57 x 10 3 /L declared by Galli et al. (2020), implying that nutrients provided was inadequate to aid production in the bone marrow, however, higher WBC suggests defence by the immune system against pathogenic or exogenous invasions.

Conclusion
It was concluded that the use of O. gratissimum leaf extract favourable compete with the conventional antibiotics on the growth performance of broiler chicken. However, extract from air-dried leaf improved survivability of the chickens.

Funding
The authors did not receive support from any organization for the submitted work. It was self-sponsored.

Competing interests
The authors declare that they have no known competing con ict of interests or personal relationships that could have appeared to in uence the work reported in this paper.

Ethics approval
The study was approved by the research ethics committee of College of Animal Science and Livestock Production of the University in line with the principle of Livestock research Consent to participate All the authors willingly participated in the research work when they were approached and their commitment to the success of the work showed that no one was coarse.

Consent for publication
All the authors gave their consent and agreed that the manuscript should be submitted to journal of Tropical Animal Health and Production for publication.
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