Evaluation of antimicrobial effect of olive leaves powder and its role in improving the broiler productivity, carcass traits, blood metabolites, and caecal microbiota

Abstract

The present study aims to evaluate the antimicrobial activity (in vitro study) of olive leaves powder (OLP) and its role in improving the broiler productivity, carcass criteria, blood indices, and antioxidant activity. A total of 270 one-day-old broiler chickens were distributed into 6 treatment groups as follows: the first group: basal diet without any supplementation, while the second, third, fourth, fifth, and sixth groups: basal diet supplemented with 50, 75, 100, 125, and 150 (µg/g), respectively. The in vitro study showed that the OLP has good antibacterial activity in the concentration-dependent matter; OLP 175 µg/mL inhibited the tested bacteria in the zones range of (0.8–4 cm), Klebsiella Pneumonaie (KP) was the most resistant bacteria to OLP concentration. The antioxidant activity of OLP increased with increasing the concentration of OLP compared to ascorbic acid, where OLP 175 µg/mL scavenged 91% of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radicals compared to 93% scavenging activity of ascorbic acid. Broiler chickens fed diets with OLP had significantly (P < 0.05) higher body weight (BW) and body weight growth (BWG) than the control birds. The treatment with OLP significantly reduced the feed intake (FI) and feed conversion rate (FCR) when compared to control. Groups supplemented with OLP showed decreased abdominal fat deposition and a significant increase in the net carcass and breast muscle weight. OLP improved birds’ blood parameters in comparison with control birds. All pathogenic bacterial numbers in caecal samples were decreased with elevating OLP levels, but the cecal Lactobacillus bacterial count was increased. In conclusion, OLP supplementation improved broiler chickens’ performance, carcass traits, and blood parameters. Moreover, OLP improved birds' liver functions (reduced Alanine transaminase [ALT] and aspartate aminotransferase [AST] levels) in comparison with control. In addition, OLP promoted the antioxidant status, minimized the harmful microbial load, and increased beneficial bacterial count in the cecal contents of broilers.

INTRODUCTION

The number of studies using natural herbs to enhance broiler chicken performance has significantly increased during the last few decades (Abd El-Hack et al., 2022a ; Rafeeq et al., 2023). The olive tree is one such plant that has attracted much attention recently (Cayan and Erener, 2015). Olive trees are wealthy in phenolic compounds with an important biological activity, the most critical of which is oleuropein (Jabri et al., 2017).

Frequent scientific investigations have evaluated the positive effects of oleuropein, olive leaf extracts (OLE) and olive leaves powder (OLP) on poultry performance (Erener et al., 2020). El-Damarawy et al. (2013) found that adding OLP to chicken ration at a level of 2.0% enhanced performance and most of the biochemical and immunological status. Abbas et al. (2012) found that adding OLP to chickens' flesh decreased feed intake and increased the feed conversion ratio. Also, Al Bandar (2017) and Xie et al., (2022) observed when supplementing olive leaves with avian ration; they enhanced the respiratory system of birds, improved the weight and egg number, enlarged the bird's weight, and enhanced the FCR.

Recently, de-Cara et al. (2023) discovered that meat with improved color intensity and fewer drip losses was produced utilizing an olive leaf and grape-based by-product. Also, Lins et al. (2018) mentioned the antioxidant activity of OLE. Several antibacterial activities of oleuropein and its related compounds have been demonstrated in vitro; in addition, because one of its metabolites, hydroxy-tyrosol, showed higher in vitro impact against different bacteria, mycoplasmas, and viruses, oleuropein could be used to produce another associated antimicrobial agent (Furneri et al., 2010).

This antioxidant nature of olive leaf extracts and antimicrobial properties is used to prolong the shelf life of food products, particularly meat manufacturing (Hayes et al., 2010). Elsaadany (2018) concluded that OLE (oleuropein) added with a level of 150 mg/kg ration, enhanced the lipid profile, performance, antioxidative, and immunological status of layer chickens. It is hypothesized that the dietary addition of OLP is expected to exert beneficial effects on growing quails. Therefore, the purpose of this study was to evaluate the antibacterial and antioxidant activity of OLP and its beneficial effects on the growth, feed utilization, carcass traits, hematology, blood constituents, and cecal microbiota of broiler chickens.

MATERIALS AND METHODS

Preparation of OLP

Dry olive leaves were purchased from a local supplier. The leaves were cleaned and converted into powder via a cereal mill. OLP was supplemented in the ratio of broilers from the first day till the end of the experiment. OLP was added to the feed of groups number 2, 3, 4, and 5 with a level of 150, 75, 100, 125, and 150 (µg/g) diet, respectively.

In Vitro Antimicrobial Activity Estimation of OLP

OLP concentrations (50, 75, 100, 125, 150, and 175 µg/mL) were performed against harmful microorganisms (Listeria monocytogensis [LM], Staphylococcus aureus [SA], Escherichia coli [EC], and Klebslia pneumonia [KP]). The tested strains were grown in Muller Hinton broth (MHB) using a shaker incubator at 37°C for 12 h for a concentration of (1 × 10⁸ CFU/mL). The disc diffusion assay performed the antibacterial impact of OLP (Ashour et al., 2020). The bacteria and Candida inoculum (100 µL) were spread over petri plates then paper discs (6 mm) saturated with OLP concentrations were located on the surface of the plate. The MHA plates were cultured for 24 h at 37°C. A ruler was utilized to calculate the inhibition zones (mm). Levofloxacin was used as a positive control to compare the antimicrobial results.

Experimental Design and Housing Condition

A total of 270 day-old broiler chickens were divided into 6 groups. Birds were allotted as follows: group 1) basal diet (G1: without any supplementation), while the other groups 2 (G2), 3 (G3), 4 (G4), 5 (G5) and 6 (G6) supplied the basal diet with 50, 75, 100, 125, 150 (µg/g) diet, respectively. The birds were housed in a semi-open clean poultry house, and then every pen was covered with clean wood shaving as bedding. The house was divided into 6 pens, each including 45 birds/pens, and each pan was sub-divided into 3 replicated 15 birds each. Rearing conditions were like all treatment groups. During the experimental period, continuous lighting was provided for 24 h in natural sunlight during the daytime and artificial lighting during the night, using bulb lamps (100 watts); they were suspended and adapted to suit the heat requirements of chicks. Ration and clean water were provided to birds ad libitum. Birds were vaccinated against avian influenza (AI) by S/C injection at the base of the neck at day old, then Newcastle disease (ND) and infectious bronchitis (IB) on the third day of age. Moreover, Gumboro disease and ND vaccines were administered in drinking water at d 16 and 21 of age, respectively.

Broiler Growth Performance

Through the 35-d experimental time, growth performance was estimated. Body weight (BW) and feed consumption were recorded weekly, and body gain and FCR were calculated. Mortality was reported during the experimental period.

Body Weight

At the beginning of the trial, each bird was weighed individually (starting BW), and then every week at 7, 14, 21, 28, and 35 d old.

Body Weight Gain

This trait was calculated by subtracting the BW of each week from the previous one. The difference was divided by the d of the week (7 d).

Feed Intake

Feed intake (FI) was computed daily, operating the weight of provided ration minus the amount of feed rejected as measured in grams per bird. Feed intake measurements were taken weekly for each pen.

Feed Conversion Ratio

Feed consumption (g) divided by weekly BW growth (g) throughout the experimental period was used to obtain the feed conversion ratio. Daily mortality checks were performed on every cage, and dead birds were removed. Feed intake and feed conversion efficiency were modified in response to mortalities.

Determination of Carcass Trait and Abdominal Fat Deposition

Three birds/replicates from each treatment were arbitrarily chosen after feeding (5 wk), weighed, and sacrificed humanely by sealing the jugular vein. The carcasses were exposed, and the liver, spleen, heart, and breast muscles were taken and weighed individually. The abdominal fat pad (fat tissues around the gizzard, bursa of Fabricius, cloaca, and neighboring muscles) was also eliminated. The following formula was used to get the proportion of the weight of the abdominal fat:

$$\frac{\text{Weight}\text{of}\text{abdominal}\text{fat}}{\text{BW}}\times 100$$

Biochemical Analysis and Blood Sampling

Blood collection was carried out during the fourth and fifth weeks of age. 5 mL blood samples were ethically harvested from the wing vein from 6 birds per replicate. Collected samples sub-divided into 3 samples were collected on anticoagulant (EDTA) coated tubes to check different blood indices. The remaining 3 samples were centrifuged for 15 min, and their serums were separated to measure levels of other metabolites.

Antioxidant Activity Against DPPH Free Radicals

The scavenging ability of OLP was revealed by Jia et al. (2012). The developed color was read at 515 nm after incubating the combination of 0.1 mL of OLP concentrations (50, 75, 100, 125, 150, and 175 µg/mL) and 3 mL of 0.1 mM ethanolic DPPH solution in the dark for 30 min. The antioxidant activity of OLP against DPPH was calculated as

$$\%\text{Scavenging}\text{activity}=\frac{\text{AC}-\text{AS}}{\text{AC}}\mathrm{x}100\left(\text{Ashour}\text{et}\text{al}.,2020\right)$$

Protein Profile Analysis

Serum was examined for the levels of TP and primary protein fractions to investigate the alterations in the protein profile within the fattening phase in chickens. Using commercially accessible diagnostic kits and an automated biochemical analyzer called Alizé (Lisabio, Poully en Auxois, France), the TP (g/L) was calculated (Randox, London, UK). According to the manufacturer's instructions, the zone electrophoresis method separated the serum protein fractions on an agarose gel using the automated electrophoresis equipment Hydrasys (Sebia Corporate, France) and commercial diagnostic equipment kits Hydragel 7 Proteine. The electrophoretic gels were scanned using the densitometry scanning equipment Epson Perfection V700 (Epson America Inc.,Los Alamitos) based on light transmission and conversion into an optical density curve. The software Phoresis version 5.50 was used to visualize the gel pictures (Sebia Corporate, Norcross, Georgia). Prealbumin, albumin, alfa1- (1-), alfa2- (2-), beta- (-), and gamma (-)-globulins were all recognized as protein fractions. According to the acquired optical density, every protein fraction was reported as relative concentrations (percent). The absolute concentrations of the fractions (g/L) were calculated from the total serum protein concentrations. By dividing the total amount of prealbumin and albumin by the total amount of globulin fractions, the albumin to globulins ratio (A/G) was obtained.

The Serum Alanine Transaminase and Aminotransferase

They were estimated utilizing commercial kits aminotransferase (AST, AS101) and alanine transaminase (ALT, AL100) – (Randox) and a spectrophotometer. AST and ALT were determined at 546 nm.

Total Lipids

Using a gravimetric measurement (Perkin-Elmer model AD-2 auto-balance) to quantify the total lipid in organic extracts, samples were diluted adequately with chloroform before being subjected to chemical analysis. Fat was isolated from plasma following the fundamental Folch et al. (1957) technique. Four milliliters of plasma were obtained for 30 min while being shaken with a 100 mL chloroform-methanol (2:1 v/v) solution, then for another 30 min while being shaken again with 20 mL 9 percent sodium chloride. The organic layer was gathered over anhydrous sodium sulphate and filtered clear after standing in a separatory flask at 4°C overnight. The proportion of total lipids was calculated using gravimetric measurement and known volumes of the organic layer.

Blood Parameters

Red blood cell (RBC) count, hemoglobin (Hb) concentration, packed cell volume (PCV), white blood cell (WBC), and differential counts were measured in the blood samples containing EDTA (Schalm et al., 1975). Within 1 to 2 h of blood collection, blood samples were examined using an automated cell counter for PCV, Hb concn, and RBC count (Celltac MEK-6108 K; Nihon-Kohdon, Tokyo, Japan). The Schalm et al. (1975) straight-edge method performed the differential leucocyte counts. A day after blood collection, plasma from centrifuged blood without EDTA was used for TP analysis. Refractometer analysis was performed on the TP.

Bacteriological Analysis of Caecal Contents

At the finale of the investigation, 1 gram of caecal content was gathered from 3 birds/ replicate to estimate total bacterial count (TBC), Total yeast and mold count (TYMC), EC count, Salmonella count, and lactic acid bacterial count (LAB). Each cecal digesta homogenate in PBS (1 mL) was serially diluted from 10⁻¹ to 10⁻⁷. Dilutions were consequently plated on duplicate selective agar media to enumerate bacterial target groups. Total aerobes bacterial count, coliforms, Salmonella, Lactobacillus spp., and TYMC enumerated using nutrient agar, MacConkey agar, Lactobacillus MRS Agar (LMRS) agar, and potato dextrose agar respectively, following Tuohy et al. (2002). Plates were then kept at 37°C for 24 h under aerobic conditions for bacteria and 5 to 7 d at 25°C for fungi, and then colonies were counted.

Statistical Analysis

The 1-way analysis of variance was employed to evaluate the data (ANOVA). Software from the statistical analysis system (SAS) was used for all analyses (version 9.0-2004). The complete randomized block design (CRBD) was employed for this investigation. A mean and standard deviation were used to describe the findings (P ≤ 0.05).

RESULTS

In Vitro Antimicrobial Activity Estimation of OLP

Figure 1 shows that OLP has good antibacterial activity in the concentration-dependent matter; OLP 175 µg/mL inhibited the tested bacteria in the zone range of (0.8–4 cm), KP was the most resistant bacteria to OLP concentration, while SA was sensitive. The MIC levels ranged from 20 to 35 µg/mL. The gram-negative microbes E. coli revealed a slightly higher sensitivity to ethanolic and water extract of green leave than the powder leave. At the same time, control discs neomycin possessed significantly higher antimicrobial action than the extracts (16 mm).

Figure 1.

Antibacterial activity of OLP against MDR-pathogenic bacteria, Antibiotic, Levofloxacin. The OLP has good antibacterial activity in concentration dependent matter, OLP 175 µg/mL inhibited the tested bacteria in the zones range of (0.8–4 cm), KP was the most resistant bacteria to OLP concentration while SA was the sensitive. The MIC levels ranged from 20 to 35 µg/mL. It is evident that the gram-negative organisms EC exhibited a slightly higher sensitivity to ethanolic, & water extract of green leave contrasted to OLP, while control discs neomycin possessed significantly higher antimicrobial action contrasted with the extracts (16mm).

Broiler Growth Performance

The effect of different concentrations of OLP dietary supplementation on the growth parameters of broiler chickens was summarized in Table 1. There was no significant difference among all the experimental groups in the initial BW (IBW). In contrast, final BW (FBW) revealed a significant (P = 0.032) difference in all groups provided with varied concentrations of OLP in comparison with the control birds (1960.1 ± 1.7). The FBW increased numerically in OLP-supplemented groups in a dose-dependent manner while there was no statistical significant difference between G3, G4, G5, and G6. The FI showed no significant difference between G2 and G1 (control group), while G3, G4, G5, and G6 led to significantly lower FI in comparison with control G1 (P = 0.019).

Table 1.

The impact of dietary OLP concentration on the growth performance of broilers from 0 to 35 d of age.

Parameters1	Control (G1)	OLP (µg/g)					P-value
		50 (G2)	75 (G3)	100 (G4)	125 (G5)	150 (G6)
IBW (g)	45.32 ± 0.6	45.36 ± 0.2	45.41 ± 0.7	45.45 ± 0.2	45.55 ± 0.2	45.68 ± 0.2	0.100
FBW (g)	1960.1 ± 1.7c	2090.69 ± 2.5b	2130.2 ± 1.3ab	2150.6 ± 1.7ab	2160.5 ± 0.7a	2166 ± 0.9a	0.032
FI (g)	3090.2 ± 3.1a	3060 ± 1.6ab	3020.2 ± 1.9c	3010 ± 2.1c	3025.8 ± 1.2b	3036 ± 1.2b	0.019
FCR (g/ g)	1.725 ± 0.1a	1.515 ± 0.9b	1.499 ± 0.5c	1.485 ± 0.5c	1.533 ± 0.5b	1.548 ± 0.1b	0.021
PI (g)	660.25 ± 1.5a	645.67 ± 0.6ab	630.13 ± 0.2b	623.23 ± 0.2b	645.64 ± 1.5ab	648 ± 0.9ab	0.026
PER	0.352 ± 0.05a	0.333 ± 0.01b	0.329 ± 0.07c	0.318 ± 0.04c	0.330 ± 0.08b	0.341 ± 0.02b	0.036
EI (kcal)	10100 ± 4.2a	9822.5 ± 3.6b	9499.3 ± 2.2c	9487.22 ± 1.5c	9555.1 ± 4.7bc	9602 ± 5.6bc	0.021
EFR	5.122 ± 0.03a	4.723 ± 0.2b	4.502 ± 0.01b	4.485 ± 0.07c	4.725 ± 0.05b	4.785 ± 0.1b	0.039

¹Data are presented mean of triplicate values ± SD, lowercase lettes (a-c) in the same row indicate signficant differences between groups at probability level of 5%. Initial body weight (IBW), final body weight (FBW), feed intake (FI), feed conversion ratio (FCR), protein intake (PI), protein efficiency ratio (PER), energy intake (EI), energy efficiency ratio (EFR).

FCR was significantly lowered in G2 (1.515 ± 0.9), G3 (1.515 ± 0.9), G4 (1.485 ± 0.5), G5 (1.533 ± 0.5) and G6 (1.548 ± 0.1) in comparison with G1 (1.725 ± 0.1) (P = 0.021). Protein intake (PI) showed no significant difference in G2, G5, and G6 compared to G1, and a significant difference was recorded in G3 and G4 (P = 0.026). The PER was significantly lowered in all experimental groups administered different levels of OLP compared to G1 (P = 0.036). Energy intake (EI) was significantly lowered in all experimental groups administered different levels of OLP compared to G1 (P = 0.021). The EFR was significantly lowered in all experimental groups administered different levels of OLP compared to G1 (P = 0.039).

Carcass Trait

The influence of supplementation of varied concentrations of OLP on the carcass trait was summarized in Table 2. There was no significant difference in the giblet weight (liver, spleen, and heart) in all experimental groups G2, G3, G4, G5, and G6 in comparison with control G1, while the net carcass weight showed a significant difference in G4 (79.5 ± 0.6) in contrast with G1 (P = 0.042). The abdominal fat was significantly lowered in G3, G4, G5, and G6 compared to G1 (P = 0.046). The breast muscle was significantly higher in G3, G4, and G6 compared to control G1 (P = 0.032).

Table 2.

Impact of varied concentrations of dietary OLP on carcass traits of broilers.

Parameters (g)	Control G1	OLP (µg/g)					P-value
		50 (G2)	75 (G3)	100 (G4)	125 (G5)	150 (G6)
Liver	1.72 ± 0.1	1.73 ± 0.2	1.75 ± 0.2	1.78 ± 0.2	1.77 ± 0.5	1.75 ± 0.2	0.20NS
Spleen	0.085 ± 0.01	0.083 ± 0.02	0.083 ± 0.02	0.084 ± 0.05	0.081 ± 0.04	0.086 ± 0.3	0.15NS
Heart	0.511 ± 0.06	0.531 ± 0.03	0.553 ± 0.05	0.577 ± 0.08	0.520 ± 0.09	0.556 ± 0.9	0.26NS
Carcass	76.2 ± 0.5b	76.8 ± 0.3b	78.2 ± 0.2ab	79.5 ± 0.6a	78.6 ± 0.9ab	78.6 ± 0.1ab	0.042
Abdominal fat	1.41 ± 0.1a	1.30 ± 0.2ab	1.20 ± 0.3b	1.11 ± 0.2bc	1.15 ± 0.6bc	1.25 ± 0.2b	0.046
Breast muscle	33.96 ± 0.6c	33.25 ± 0.4c	38.99 ± 0.7ab	40.23 ± 0.6a	34.65 ± 0.4c	35.11 ± 0.6b	0.032

NS= Nonsignificant, Results are offered mean ± SD, P-value ≤0.05 indicate significant difference.

Biochemical Analysis and Blood Analysis

Figure 2 shows that OLP's antioxidant activity increased with concentration compared to ascorbic acid, where OLP 175 µg/mL scavenged 91% of DPPH radicals compared to 93% scavenging activity of ascorbic acid. The SC50 of OLP was 50 µg/mL.

Figure 2.

Antioxidant activity of OLP against DPPH free radicals; the antioxidant activity of OLP elevated with concentration increased compared to ascorbic acid, where OLP 175 µg/mL scavenged 91% of DPPH radicals compared to 93% scavenging activity of ascorbic acid (P > 0.05). the SC50 of OLP was 50 µg/mL.

The effect of dietary OLP different concentrations on broiler's hematological and serum biochemical parameters at 35 d of age was shown in Table 3. Regarding the serum biochemical parameters, total protein, albumin, and globulin, there was no significant difference among all groups treated with different levels of OLP in contrast with G1. The albumin/globulin ratio showed a significant difference in G2, G3, G5, and G6, no significant difference was detected in G4 compared with G1 (P = 0.032). Total lipids were significantly lowered in all groups with varied levels of OLP contrasted to G1 (P = 0.020). Serum ALT revealed a significant decrease in all experimental groups with varying levels of OLP compared to G1 (P = 0.016). Serum AST showed a significant decrease in all experimental groups treated with different levels of OLP in contrast with G1 (P = 0.012).

Table 3.

Effect of dietary OLP concentrations on hematological and serum biochemical parameters of broiler at 35 d old.

Parameters1	Control (G1)	OLP (µg/g)					P-value
		50 (G2)	75 (G3)	100 (G4)	125 (G5)	150 (G6)
Hematological parameters
RBCs (106/µL)	2.41 ± 0.1c	2.45 ± 0.2c	2.65 ± 0.3b	2.71 ± 0.5a	2.62 ± 0.2b	2.63 ± 0.1b	0.05NS
HGB (g/dL)	12.1 ± 0.2c	12.3 ± 0.2c	12.8 ± 0.2b	13.5 ± 0.2a	12.7 ± 0.3b	12.9 ± 0.2b	0.09NS
HCT (%)	24.9 ± 0.3c	25.3 ± 0.1c	26.55 ± 0.3b	27.66 ± 0.0a	27.00 ± 0.5ab	27.12 ± 0.3ab	0.07NS
MCV (µm3/ cell)	105.5 ± 0.2a	103.9 ± 0.3c	102.78 ± 1.4c	100.68 ± 0.6d	104.8 ± 0.3ab	103.8 ± 0.2b	0.041
MCH (g/dL)	52.3 ± 0.1a	51.3 ± 0.5ab	47.77 ± 0.3c	47.68 ± 0.5c	48.33 ± 0.6b	48.85 ± 0.5b	0.042
MCHC (g/dL)	47.12 ± 0.3a	46.88 ± 0.6ab	46.12 ± 0.9b	45.56 ± 0.2c	46.2 ± 0.9b	46.35 ± 0.3b	0.1NS
PLT (104/ µL)	39.21 ± 1.2a	37.45 ± 0.3b	32.52 ± 1.3d	32.77 ± 0.4d	33.51 ± 2.3c	33.78 ± 0.9c	0.045
WBCs (102/µL)	135.0 ± 1.3a	129.23 ± 0.1b	127.75 ± 0.8c	127.63 ± 1.1c	128.55 ± 2.2bc	128.98 ± 1.2bc	0.032
Serum biochemical parameters
Total protein (g/ dL)	2.75 ± 0.2	2.78 ± 0.2	2.88 ± 0.3	2.99 ± 0.3	2.89 ± 0.2	2.90 ± 0.2	0.07NS
Albumin (g/ dL)	1.32 ± 0.3	1.38 ± 0.5	1.43 ± 0.5	1.45 ± 0.2	1.48 ± 0.9	1.51 ± 0.3	0.08NS
Globulin (g/ dL)	1.29 ± 0.1	1.33 ± 0.4	1.38 ± 0.1	1.42 ± 0.7	1.38 ± 0.2	1.39 ± 0.5	0.095NS
A/G ratio	1.02 ± 0.1c	1.03 ± 0.6b	1.03 ± 0.2b	1.02 ± 0.5c	1.07 ± 0.5a	1.08 ± 0.6a	0.032
Total lipids (mg/ dL)	420.67 ± 1.7a	400.2 ± 0.1b	392.0 ± 2.5c	379.23 ± 2.2e	381.67 ± 3.1d	385 ± 1.2d	0.020
ALT (U/ L)	17.19 ± 0.0a	10.6 ± 0.3b	9.98 ± 0.9bc	9.88 ± 0.3bc	10.2 ± 0.2b	10.9 ± 0.3b	0.016
AST (U/ L)	58.33 ± 0.2a	45.5 ± 0.5b	39.01 ± 0.8c	38.55 ± 0.6d	39.68 ± 0.4c	39.77 ± 0.5c	0.012

NS= Nonsignificant, Results are displayed mean ± SD, P-value ≤0.05 indicate significant difference.

¹Data are presented as mean of triplicate values; lowercase letters (a-c) in the same row indicate significant differences at p<0.05. Red blood cell (RBC) count, haemoglobin (HGB) concentration, hematocrit (HCT); packed cell volume (PCV), white blood cell (WBC), platelets (PLT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV); alanine transaminase (ALT) and aspartate aminotransferase (AST).

The hematological blood analysis revealed that RBCs (10⁶/µL), Hb (g/dL), hematocrit (HCT) (%) and mean corpuscular hemoglobin concentration (MCHC) (g/dL) showed no statistically significant difference among all the treated groups in comparison with G1. Mean corpuscular volume (MCV) (µm3/ cell) revealed a significant decrease in G2, G3, G4, and G6 in comparison with G1 (P = 0.041). Mean corpuscular hemoglobin (MCH) (g/dL) showed a significant decrease in G3, G4, G5, and G6 in contrast to G1 (P = 0.042). The PLT (104/ µL) showed a significant decrease in all groups provided with varied concentrations of OLP contrasted to G1 (P = 0.045). The WBCs (102/µL) revealed a significant decrease in G2, G3, G4, G5, and G6 in contrast to G1 (P = 0.032). The significant decrease in WBCs count in OLP-treated groups indicates its effect on limiting pathogens.

Bacteriological Analysis of Caecal Contents

The influence of supplementation of varied concentrations of OLP on birds’ ratio on the caecal microbial count at 35 d of bird age was summarized in Table 4. The TBC was significant lowered in a dose dependent manner in G2 (4.3 ± 0.1), G3 (2.9 ± 0.5), G4 (2.1 ± 0.3), G5 (1.5 ± 0.1) and G6 (0.9 ± 0.3) in comparison with G1 (7.7 ± 0.2) (P = 0.0156). The TYMC was significant decrease in a dose dependent manner in G2 (3.6 ± 0.1), G3 (3.0 ± 0.2), G4 (2.2 ± 0.1), G5 (1.8 ± 03) and G6 (1.0 ± 0.0) in comparison with G1 (5.2 ± 0.2) at (P = 0.0265). E. coli caecal count revealed that G6 showed no E. coli growth, indicating that the OLP at a concentration of 150 (µg/g) could stop E. coli multiplication. Also, all the OLP-treated groups showed a significant decrease in E. coli count compared to G1 (P = 0.0148). Salmonella was only detected in control birds, while not in all the experimental groups treated with different concentrations of OLP (P < 0.001). The LAB count showed a significant (P < 0.001) increase in all OLP treated groups G2 (4.5 ± 0.2), G3 (5.9 ± 0.6), G4 (7.1 ± 0.4), G5 (6.2 ± 0.5), and G6 (7.2 ± 0.3) in contrast to G1 (2.5 ± 0.1). In conclusion, different levels of OLP succeeded in reducing pathogens and increasing beneficial bacteria.

Table 4.

Microbial count in caecal contents of broiler fed on diet supplemented with OLP at different concentration after 5 wk feeding time.

Concentration (µg/g)	Microbial count1
	TBC	TYMC	E. coli	Salmonella	LAB
Control (G1)	7.7 ± 0.2a	5.2 ± 0.2a	3.1 ± 0.2a	0.25 ± 0.05	2.5 ± 0.1e
50 (G2)	4.3 ± 0.1b	3.6 ± 0.1b	2.1 ± 0.1b	ND	4.5 ± 0.2d
75 (G3)	2.9 ± 0.5c	3.0 ± 0.2bc	1.8 ± 0.2bc	ND	5.9 ± 0.6c
100 (G4)	2.1 ± 0.3d	2.2 ± 0.1c	0.9 ± 0.05c	ND	7.1 ± 0.4a
125 (G5)	1.5 ± 0.1de	1.8 ± 03cd	0.3 ± 0.01d	ND	6.2 ± 0.5b
150 (G6)	0.9 ± 0.3e	1.0 ± 0.0d	ND	ND	7.2 ± 0.3a
P value	0.0156	0.0265	0.0148	0.001	0.001

ND= Not detected, Results are displayed mean ± SD, P-value ≤0.05 indicate significant difference.

¹Lowercase letters (a-d) in the same column indicate significant differences at p<0.05. TBC (total bacterial count), TYMC (total yeast & mold count), and LAB (lactic acid bacterial count).

DISCUSSION

The risk of multiple drug resistance in human pathogenic bacteria and the resulting cross-resistance are increased by the excessive use of antibiotics in modern chicken production (Abd El-Hack et al., 2022a). To protect the health and welfare of animals and lessen the industry's negative environmental effects, the European Union restricted adding a wide range of antibiotics in chicken diets (Swelum et al., 2021; El-Saadony et al., 2022; Saleh et al., 2023).

Recently, there has been an elevation in the use of natural products as feed additives (herbs, plant extracts, and essential oils) and bio-growth promoters (Abd El-Hack et al., 2022b; El-Saadony et al., 2023). Olive leaves contain many phenolic compounds (Agah et al., 2019; Bilal et al., 2021). Oleuropein is the prevalent phenolic structure in olive leaves (Debbou-Iouknane et al., 2021). Analgesic, anti-inflammatory, antioxidant, antibacterial, antitumor, and anticancer properties are only a few of the biological effects that the phenolic materials in olive leaves have been displayed to have in earlier studies (Oke et al., 2017).

Klebsiella pneumoniae (KP) was the most resistant bacteria to OLP concentrations in this study, which examined the antibacterial activity in a concentration-dependent manner. OLP 175 g/mL inhibited the tested bacteria in the zone range of (0.8–4 cm). Staph aureus (SA) was sensitive at the same time. The extract’s high concentration of phenolic and flavonoid components may be a factor in its antibacterial abilities. It was claimed that phenolic compounds' antimicrobial effects were linked to the deactivation of cellular enzymes, which was influenced by the substance's rate of cell penetration or brought on by modifications in membrane permeability (Selim et al., 2022).

In the mechanism of antimicrobial activity, increased membrane permeability is a crucial component. Compounds may upset membranes, resulting in a loss of cellular integrity and eventual cell killing (Selim et al., 2022). According to the results of the current study, broiler feed containing OLP considerably enhanced BW and BWG while decreasing FI and FCR. Our results are consistent with those of El-Damarawy et al. (2013), who found that adding OLP (0.5, 1.0, and 2.0 percent) to the Mandarah chick meal significantly enhanced daily weight gain and BW. A study found that broilers provided ration with 100 and 200 mg kg⁻¹ OLE gained more BW and had a more excellent FCR than the control birds (Sarıca and Ürkmez, 2016 ).

According to Younan et al. (2019), OLE had negligible effects on all groups' feed consumption. The best FCR and improved rabbit performance were evident over the trial period in a considerable rise in average daily WG. With the addition of aqueous olive leaves extract (AOLE), the average daily gain (ADG) and FCR of the birds increased significantly throughout the experiment's necessary time (Jabri et al., 2017). Additionally, Oke et al. (2017) discovered that adding OLE to broiler chicks' drinking water dramatically improved FBW, overall WG, and FCR. The addition of oleuropein (400 ppm) to the diet of Japanese quail improved the feed conversion ratio (Bahsi et al., 2016).

Adding various OLP doses did not change goblets weight in this trial. Still, it dramatically raised final net BW and breast muscle weight while decreasing the percentage of abdominal body fat accumulation. Due to alterations in lipase and bile acid secretions caused by plant antioxidant components, belly fat depositions would reduce compared to the control (Amini et al., 2019). It has been documented that olive derivatives have hypolipidemic effects. According to Sarıca and Ürkmez (2016), oleuropein, an antioxidant found in olive leaves, inhibits the activity of several enzymes involved in synthesizing lipids in the liver, including hydroxymethylglutaryl-CoA synthase and hydroxyl-methylglutaryl-CoA reductase. Flavonoids in food reduce phosphodiesterase activity and fat tissue synthesis (Rakha et al., 2022 ).

The data of this study were regular with those of earlier research by Shafey et al. (2013), which found that broiler chickens' belly fat content was reduced when olive leaves were given to them. According to our findings on abdominal fat, the reduction in abdominal fat and the percentages of meat fat influenced by OLE treatments were noted by Younan et al. (2019). As a result of a fast development rate associated with greater body fat deposition, Zubair and Leeson's (1996) findings that higher belly fat may be caused by it until slaughtering age disagreed with the results of the current investigation. Therefore, it can be said that high OLE-supplemented diets have a disadvantage when it comes to having increased body fat levels. The yield of head, heart, gizzard and neck, as well as abdominal fat, did not differ (P > 0.05) in broilers supplied varied levels of an olive meal with or without the inclusion of the enzyme supplement, according to Sateri et al. (2017) and Al-Harthi (2017) findings. Additionally, Erener et al. (2020) discovered that the weight of control broilers' abdominal fat was lower than that of all OLE birds.

According to our findings, OLP's antioxidant activity rose with concentration compared to ascorbic acid; at 175 g/mL, OLP scavenged 91 percent of DPPH radicals compared to ascorbic acid's 93 percent scavenging activity. OLP has a SC50 of 50 g/mL. OLP contains various substances collectively referred to as olive bio-phenols, which are contemplated to be responsible for the extract's varied medicinal qualities. Oleuropein, the primary ingredient in olive leaf extract, is antibacterial, cardioprotective, antiviral, antioxidative, antiatherogenic, and antihypertensive. Oleuropein is a member of the secoiridoids class of coumarin-like substances. This compound has many Oleaceas, Cornales, Gentianales, and other herbs. Typically, glycosidic bonds bind iridoids and secoiridoids (Zheng et al., 2022). They are created from terpenes' secondary metabolism as a source of several indole alkaloids (Zheng et al., 2022).

One of the iridoid monoterpenes is oleuropein, a (3,4-dihydroxyphenyl) ethanol (hydroxytyrosol) ester with -glucosylated oleanolic acid. Oleuropein oversees the therapeutic actions (Nenadis et al., 2023). Oleuropein's main biological impacts have been shown to include its anti-inflammatory and antioxidant effects also its ability to treat oxidative and inflammatory disorders such as diabetes, obesity, hepatitis, and cardiovascular disease. Oleuropein suppresses the growth of various tumor cell types and exhibits anticlastogenic and free-radical scavenging effects. This phenol can prevent lipoxygenases and low-density lipoprotein oxidation. It also has hypocholesterolemic and hypoglycemic effects. It is also recognized for facilitating obesity issues by enhancing lipid metabolism, as stated in various research (Nenadis et al., 2023).

The alteration of physiological indices can affect characteristics related to the health of birds (Arif et al., 2022). According to the current study, OLP improved lipid profiles, ALT or AST levels, and blood parameters compared to the control. The beneficial effects of olive extracts on the liver functions of chickens were validated in a concurrent investigation by Tavakolinasab et al. (2020). Additionally, Agah et al. (2019) discovered that chickens fed diets containing olive leaf extract had considerably lower blood levels of cholesterol, triglycerides, ALT, and AST and lower plasma lipid peroxidation levels, and lower glutathione peroxidase activities.

In this investigation, OLP considerably raised the healthy bacteria count and decreased the number of caecal pathogens. These data were in concur with those of Jabri et al. (2017), who discovered that an aqueous olive leaf extract with a dose of 10 mL/L had the greatest effective activity (P-value 0.05) against cecal pathogenic bacteria. Additionally, it considerably promotes Lactobacillus growth.

CONCLUSIONS

OLP has in vitro antibacterial activity, and the data of this experiment reveal that the use of OLP as natural feed additives in broiler chickens diets, especially with the rate of 100, 150 µg/g feed has a beneficial impact on the performance, carcass trait, blood parameters, liver function, antioxidant capacity, lipid profile, abdominal fat deposition, caecal microbial count.