Effect of olive leaf (Olea europaea L.) extract addition to broiler diets on the growth performance, breast meat quality, antioxidant capacity and caecal bacterial populations

Abstract

This study aims to evaluate the effect of olive leaf extract (OLE) on growth performance, breast meat quality, antioxidant capacity and caecal microbiota regulation. OLE containing 51.5% oleuropein, 25.6% polyphenols and 6.2% flavonoids, was designed to different supplement levels ranging from 0.1% OLE (OLE0.1) to 0.5% OLE (OLE0.5) into a basal diet, separately. The whole feeding period lasts for 35 days after 7-day hatching from the egg. 720 1-day-age healthy Arbour Acres male broilers were thus divided into five experimental groups and one control group, consist of 20 broilers per group for six parallel experiments in triplicate. The results showed that the levels of average daily feed intake (ADFI) and average daily weight gain (ADG) of the broilers were decreased (p < 0.05) after the addition of 0.3% OLE. Moreover, the breast meat shear force was decreased by 34.4% (p < 0.05). The glutathione (GSH) levels in the meat were increased by 76.64% (p < 0.05); the levels of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) were increased by 50.4% and 120%, respectively. And the malondialdehyde (MDA) levels in the meat were reduced by 34.2% (p < 0.05). Additionally, a decreased intensity of Escherichia coli (p < 0.05) and an increased intensity of Lactobacillus and Bifidobacterium (p < 0.05) were observed. Overall, supplementing 0.3% OLE into daily diet enables enhancing breast meat antioxidation, amplifying the abundance of Lactobacillus and Bifidobacterium as well as diminishing the abundance of E. coli in the caecum.

HIGHLIGHTS

• OLE contains 51.5% oleuropein, 25.6% of TP and 6.2% TF levels.

• OLE addition at 0.3% lowers shear force and raises the GSH’s level of the meat.

• OLE causes a decrease in E. coli and an increase in Lactobacillus and Bifidobacterium.

Graphical Abstract

Keywords:

• Oleuropein
• shear force
• GSH and MDA
• microbiota intensity
• meat quality

Introduction

In 2021, 135 million tons of chicken meat were produced worldwide, which reached approximately 40.4% of the total production of livestock and poultry meat (Shahbandeh 2022). Oxidative stress in the industrialised breeding process is yielded and production performance, health and meat quality in chicken birds are severely affected (Sola-ojo et al. 2019). Antioxidants are vital tools to counteract these detrimental impacts. Among them, synthetic antioxidants such as BHT, BHA and propyl gallate applied in animal breeding could work, however, are not confirmed to be safe for animals even human beings due to their increased incidence of malignant tumour (Felter et al. 2021). Conversely, natural antioxidants applied in poultry are well recognised to be safe, effective, reliable and wide-sourced. Therefore, seeking natural effective antioxidants from agri-forest residues to lower oxidative stress and improve chicken meat quality, is very pivotal.

Olive leaf (Olea europaea L.), as byproducts of 330,000 tons/year around the world, is one kind of potential animal nutrient used for high-quality meat. The increasingly high demand for green and healthy husbandry using natural polyphenols is available (Achat et al. 2012). However, olive leaf is abundant in crude fibre (over 26%), which may not be suitable for directly feeding chickens, owing to the difficulty in digestion and palatability (Altop et al. 2018). Oleuropein dominates the olive leaf with a variety of ranging from 8% to 14% (Japon-Lujan and de Castro 2006). Flavonoids (luteolin, apigenin and rutin, etc.) and single phenols (vanillin, caffeic acid, tyrosol, hydroxytyrosol, etc.) are also present in olive leaf (Rahmanian et al. 2015). Olive leaf extract (OLE) displayed a stronger antioxidation than vitamins C and E due to the synergic performance between oleuropeosides and other bioactive phenols (Benavente-Garcı́a et al. 2000). Oleuropein shows plentiful functions, for example, antioxidation and anti-inflammation (Hassen et al. 2015). The safety of OLE as a sensory additive in feed for all animal species was confirmed by the European Food Safety Authority (Authority EFS 2007). Different studies into feed additives of poultry for meat quality enhancement have concentrated on natural plant (extracts) for the sake of safety and effectiveness (Koné et al. 2019; Paiva et al. 2021). Previous findings showed that broiler meat quality and growth performance of chicken are associated with caecal microbiota (Zhang et al. 2021a, 2021b). Dried OLE can be thereby used as an effective additive for improving growth performance and antioxidation in vivo for poultry industry. Additionally, olive byproducts such as olive cake and other phytogenic leaves or extracts (i.e. Moringa oleifera) were also widely applied in poultry owing to the abundant level of phenolic compounds resulting in notably improved growth performance and meat quality as well as health status (Saleh et al. 2014, 2020; Saleh and Alzawqari 2021; Selim et al. 2021).

Taken together, olive leaf (extract) is a cheap and abundant source of phenolic compounds and is now widely used in functional foods and husbandry fields, such as ruminant animals (sheep, goat and cow) or rabbits (Molina-Alcaide and Yáñez-Ruiz 2008; Ribeiro et al. 2012). However, data on the effectiveness of OLE supplemented into the corn-soybean-based diets of broiler chickens for improving meat quality, to the best of our knowledge, are still limited. Therefore, this study aims to evaluate the effect of OLE on growth performance, breast muscle meat quality, antioxidant capacity and caecal microbiota regulation. All the findings indicate OLE can be potentially a feed additive in poultry due to its enhancing breast meat antioxidation and caecal microbial regulation.

Materials and methods

All procedures concerning the broiler chickens were performed in compliance with relevant laws and institutional guidelines (Animal Care and Use Guidelines), and these experiments obtained approval (China, NAU-2018320122000011) from the Animal Care and Use Committee of Nanjing Agricultural University.

Materials

Dried OLE (in an oven at 50 °C for 48 h) was purchased from Tianyuan Olive Development Inc. (Longnan, China). The OLE was extracted by the following protocol. A certain amount (1.0 kg) of olive leaf (Longnan City, Gansu Province), dried to approximately 4.6% moisture under the sun, was mixed with 75% ethanol-water in a solid–liquid ratio of 1:30 (kg/L). The resulting mixture was placed into an extraction kettle (RY-NSG-L, Shanghai RuiYuan Equipment Co. Ltd., China.) for extracting bioactive phenolic compounds from olive leaf under the status of solvent boiling at 90 °C reflux for 5 h. The extracted solution was obtained by vacuum filtration and then evaporated to remove ethanol at 50 °C. The final product of OLE was acquired by vacuum freezing for 50 h. Oleuropein, gallic acid and rutin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Acetonitrile and formic acid were purchased from Tedia Inc. (Fairfield, OH, USA). All chemicals were at least reagent grade.

Analyses of phenolic compounds in olive leaf extract by high performance liquid chromatography-microelectrospray ionisation time-of-flight mass spectrometry

The characterisation of OLE was implemented by a Shimadzu LC20 liquid chromatography system (Shimadzu Technologies, Tokyo, Japan) with an autosampler, according to a previous report (Quirantes-Piné et al. 2013). Briefly, an HPLC column called Waters XBridge (3.5 μm, 150 × 2.1 mm) was applied to separate the phenolic compounds under the conditions of an injection volume of 5 μL, measurement wavelength of 280 nm and flow rate of 0.2 mL/min using a gradient elution program at 25 °C. The mobile phases contained acetonitrile (A) and 0.1% formic acid in water (B). The successive multistep linear gradient was required as follows: 0 min, 85% B; 5 min, 85% B; 25 min, 70% B; 35 min, 5% B; 40 min, 5% B. The primary states were kept for 5 min to adjust the HPLC baseline. Additionally, the HPLC system was equipped with a SCIEX triple microTOF mass spectrometer (Bruker Daltoniks, Bremen, Germany) connected to an ESI interface performing in negative ionisation mode. The other optimal settings of the ESI-QTOF-MS/MS parameters were drying gas temperature of 5000 °C, drying gas flow of 10 L/h, nebulising gas pressure of 2 bars, capillary voltage of 700 V and pulser frequency of 18.1 kHz. Detection was implemented in a mass range from 50 to 1100 m/z. The MS/MS analyses were acquired by automatic fragmentation in which the three most intense mass peaks were fragmented.

Total polyphenol and total flavonoid content

Total polyphenol and total flavonoid content in OLE were measured by Folin–Ciocalteu’s method under 760 nm and AlNO₃ colourant under 420 nm based on our prior protocol (Xie et al. 2015).

Care and use of broiler chicken

Feeding treatments

Seven hundred and twenty 1-day-old healthy Arbour Acres broiler male chickens with no significant weight differences were obtained from Shengnong Hatchery (Huaian, Jiangsu Province, China) and randomly designated to six treatments. 20 chicks were executed in six parallel triplicates for each treatment. The chicks with six treatments were classified as one control group fed basal diets (ingredients and nutrient content seen in Table 1), and five test groups including 0.1% OLE (OLE0.1, 1.0 g/kg diet), 0.2% OLE (OLE0.2, 2.0 g/kg diet), 0.3% OLE (OLE0.3, 3.0 g/kg diet), 0.4% OLE (OLE0.4, 4.0 g/kg diet) and 0.5% OLE (OLE0.5, 5.0 g/kg diet) mingled with the basal diets. Commercial feed based on corn-soybean came from Kangxin Hatchery (Nanjing, China). The resulting feed form of broiler chickens was mash feed.

Table 1. Composition and nutrient level of the corn-soybean basal diets.

Ingredients	0 ∼ 21 days	22 ∼ 35 days
Corn, g	57.02	61.36
Soybean meal, g	31.30	28.30
Corn gluten meal, g	3.70	1.70
The crude protein ratio of soybean meal and corn gluten meal	9.28	18.27
Soybean oil, g	3	4
Dicalcium phosphate, g	2	1.60
Limestone, g	1.20	1.30
L-Lysine, g	0.33	0.31
DL-Methionine, g	0.15	0.13
Salt, g	0.30	0.30
Calculated nutrient levels
Metabolisable energy, MJ/kg	12.57	12.91
Crude protein, %	21.42	19.23
Lysine, %	1.20	1.10
Methionine, %	0.50	0.44
Calcium, %	1	0.93
Available phosphorus, %	0.46	0.39
Premix^a	1	1

^aProvided per kg of diet: vitamin A,10,000 U; vitamin D₃, 3000 U; vitamin E, 30 U; vitamin K₃, 1.3 mg; thiamine, 2.2 mg; riboflavin, 8.0 mg; niacin, 40.0 mg; choline chloride, 600 mg; calcium pantothenate, 10.0 mg; pyridoxine, 4.0 mg; biotin, 0.04 mg; folic acid, 1.0 mg; vitamin B₁₂, 0.013 mg; zinc, 65.0 mg; iron, 80.0 mg; copper, 8.0 mg; manganese, 110 mg; iodine, 1.1 mg; selenium, 0.3 mg.

Husbandry

Before implementing experiments, the birdhouse and related appliances were completely washed with running water, disinfected with a spraying caustic soda, and then fumigated by a mixture (30:1, mL/mg) of formalin and potassium permanganate for 12 h, ultimately continuously sanitising for 1 week. 720 1-day-old healthy Arbour Acres broilers were randomly divided into 6 treatment groups (5 experimental groups and 1 control group), consisting of 20 broilers per group in six parallel experiments. The chicks were caged in wire with a three-level battery and inhabited a room controlled at 32 ∼ 34 °C from 1 to 14 days. Then, the temperature was lowered by 2 °C every week until reaching 26 °C and remained constant until the experimental termination. In this study, each cage was locked and measured 320 cm in length, 120 cm in width and 50 cm in height, and it was equipped with one tube feeder. The tube feeder with a diameter of 33 cm had 7.5-cm-wide feeder slots. They were separated equally into 2 sides, and birds on the same sides were capable of making visual contact by the wire fences. These chicks were kept in a 12 h photoperiod and 12 h darkness (12 L:12 D) based on previous findings that showed benefits in achieving the maximised feed conversion efficiency, reducing protein denaturation and improving lipid stability (Lewis et al. 2009; Tuell et al. 2020). And The chicks were vaccinated against the infectious bursal disease and Newcastle disease. Chickens could be free access to feed and water ad libitum throughout the feeding period of 35 days. The average daily gain (ADG, g), the average daily feed intake (ADFI) and the feed conversion ratio (FCR) of these chickens was measured.

Sampling and treatment of broiler chicken

After 35 days of consuming the experimental diets, 72 randomly selected broilers (2 chickens per replicate) were euthanised by cervical dislocation after blood collection, and then necropsy was executed based on the operating procedure of poultry welfare slaughtering from China (GB/T 19478-2018) and European Directive 2010/63/EU. It required a 12 h starving period before the slaughtering. The venous blood sampling was shortly implemented at the neck. The blood was placed at 4 °C until the resulting serum was completely precipitated. The sampling serum was obtained by centrifugation at 3500 x g at 4 °C for 10 min. The same 6 parts of the breast muscle meat samples were generated when the carcases were pruned by eliminating feathers, bones and connective tissues. After pruning, breast muscle meat samples were cut into different slice segments. One segment was vacuum-packed and stored at −20 °C until the next analysis of cooking loss, shear force and flesh colour. The other segments of the breast muscle meats were used for preparing 10% tissue homogenate (tissue: physiological saline = 1:9, w/w) and stored at −20 °C for further antioxidation assessment.

Storage procedure of broiler chicken samples

To evaluate the effects of different levels of OLE in the basal diets on breast muscle meat quality, antioxidant capacity and enzymes and serum biochemical indexes in chickens during refrigeration storage, the sampled breast muscle meat was enveloped in sealed plastic bags and placed at −20 °C until next use.

Cooking loss of the breast muscle meat

Samples of muscle meat without connective tissue were packed in plastic bags at 0 ∼ 4 °C for 24 h. After that, the meat was weighed and recorded as W4. The meat was placed in a water bath controlled at 75 ∼ 80 °C until the temperature in the central district of the meat reached 70 °C. The cooked meat was immediately cooled with running water, dried with filter paper and weighed (W5). Therefore, the cooking loss of the meat was estimated by the following equation: $$\text{Cooking loss }\left(\mathrm{\%}\right)=\frac{\mathrm{W}4 - \mathrm{W}5}{\mathrm{W}4} \times 100\%$$

The shear force of the breast muscle meat

Breast muscle meat sealed in the plastic bag was cooked until the meat centre achieved 74 °C in the water bath controlled at 80 °C. Each piece of cooked meat was trimmed along the muscle fibre to a size of 1 cm $\times$ 1 cm $\times$ 3 cm, which was measured by a Salter Shear Force Instrument (G2R Elec. Mfg. Co.) in triplicate.

Meat colour measurement

The sampled breast meat stored at 4 °C in the sealed plastic bag was removed from the refrigerator after 24 h, and a fresh cross-section was cut to measure the lightness (L*), redness (a*) and yellowness (b*) in the room with normal light (not exposed to direct sunlight or darkness) by a colorimeter (WSC-S, Shanghai Minyi Electronics Co., Ltd., China) with CIELAB system.

The biochemical index in the serum

Some biochemical indexes, for example, total protein, albumin, urea nitrogen, total cholesterol, triglyceride (TG), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, creatinine, alkaline phosphatase, total bilirubin, alanine transaminase (ALT) and aspartate aminotransferase (AST) were determined in the chicken serum by a kit (Nanjing Institute of Jiancheng Bioengineering, China) and all the measuring procedures were carried out in strict accordance with the protocols instructed in the kit instructions.

Antioxidant capacity of the breast muscle meat and serum in chickens

Some antioxidant biochemical indexes, for example, total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and malondialdehyde (MDA) were measured in the breast muscle meat and serum of the chickens by a kit (Nanjing Institute of Jiancheng Bioengineering, China). All measuring procedures were carried out in strict accordance with the methods in the kit instructions (http://www.njjcbio.com/).

PCR analysis of intestinal bacteria in the caeca

DNA in broiler caeca content was extracted according to a commercial Genomic DNA Extraction Kit with a centrifugal column type (Tiangen BioChem Technology Corporation, Beijing, China), using an ultra-micro spectrophotometer NanoDrop ND-2000 (Wilmington, DE, USA) to detect the purity and concentration of the DNA. The integrity of DNA was checked by 2% agarose gel electrophoresis. The obtained DNA was stored at −20 °C for next use.

The primers for Lactobacillus, Escherichia coli, Bifidobacterium group and total bacteria in caeca were designed by some previous reports (Rinttilä et al. 2004; Jensen et al. 2011). All primers were synthesised by Invitrogen Company (Shanghai, China). The details of the primer sequences, their annealing temperatures and product size (bp) are shown in Table S1. Quantification of bacterial groups of caeca was implemented by SYBR Green PCR Master Mix (Dalian Bao Bioengineering Co., Ltd.). Calibration of a series of PCR products with known copy numbers per ng DNA was executed to calculate copy numbers/g sample. An ABI real-time PCR instrument StepOnePlus™ (Applied Biosystems, USA) was used to quantitatively detect the target gene transcripts. According to the Ct value obtained, the standard curve was established and the regression equation in the corresponding linear range was obtained (see Table S2).

Table 2. Major compounds identified by HPLC-MS/MS in olive leaf extract.

Peak	R.T., min	Measured, m/z	Formula	Fragments	Proposed compounds	Content, mg/g	Mass content percent, %
1	8.797	153.0554	C8H9O3	123.0455 (100)	Hydroxytyrosol	7.68	0.768
2	22.752	623.1981	C29H36O15	133.0293 (13.8), 161.0249 (100), 461.1680 (20.7)	Verbascoside	17.14	1.714
3	23.950	447.0973	C21H19O10	285.0408 (100)	Luteotin-7-glucose	65.20	6.520
4	24.400	609.1497	C27H29O16	151.0030 (4.3), 178.9964 (4.5), 300.0264 (100)	Rutin	1.23	0.123
5	26.010	431.0973	C21H19O10	263.0380 (100)	Apigenin-7-glucose	41.94	4.194
6	26.750	541.1927	C25H34O13	121.0665 (20.0), 181.0872 (100), 193.0507 (34.3), 225.0342 (74.3), 361.1304 (72.9)	Oleuropein	515.00	51.500
7	27.300	539.1770	C25H31O13	139.0395 (24.2), 149.0254 (70.6), 223.0621 (35.3), 307.0839 (100), 377.1263 (70.6), 403.1263 (14.7)	Oleuropein isomer	80.21	8.021
8	28.510	285.0418	C15H9O6	133.0295 (7.3), 151.0013 (6.2), 175.0387 (5.2), 255.0299 (7.7)	Luteotin	61.70	6.170

R.T. indicates the retention time of the phenolic compounds identified by HPLC-MS/MS.

Statistical analysis

All data in this study concerning statistical analysis were obtained using SPSS v.18.0 software program (SPSS Inc., Chicago, IL, USA) and expressed as the means ± standard deviation. Statistically significant differences between two parameters were determined by one-way analysis of variance (ANOVA). Difference (p < 0.05) among the various treatments was implemented by Duncan’s multiple range test. The definition of tendencies indicates the value becomes significantly increased or decreased with the addition of OLE ranging from 0.1% to 0.5% as a whole.

Results

Characterisation of olive leaf extract by high performance liquid chromatography-microelectrospray ionisation time-of-flight mass spectrometry

As Figure 1 and Table 2 indicated, 25.6% of the total polyphenol content and 6.2% of the total flavonoid content in OLE were observed. The major phenolic compounds including hydroxytyrosol (7.68 mg/g), verbascoside (17.14 mg/g), luteotin-7-glucose (65.2 mg/g), rutin (1.23 mg/g), apigenin-7-glucose (41.94 mg/g), oleuropein (515.0 mg/g), oleuropein isomer (80.21 mg/g) and luteolin (61.7 mg/g), were revealed by high performance liquid chromatography-microelectrospray ionisation time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). Among them, oleuropein was concentrated at 51.5% in OLE of dry matter, followed by 6.52% luteotin, 6.17% luteotin-7-glucose and 4.194% apigenin-7-glucose.

Figure 1. HPLC profile of olive leaf extract with a concentration of 4.74 mg/mL dissolved in methanol; HPLC column of waters XBridge (3.5 μm, 150 × 2.1 mm), was applied to separate the phenolic compounds under the conditions of injection volume 5 μL, measurement wavelengths 280 nm and flow rate 0.2 mL/min using a gradient elution program at 25 °C. The mobile phases consist of acetonitrile (A) and 0.1% formic acid in water (B). The successive multistep linear gradient was required as follows: 0 min, 85% B; 5 min, 85% B; 25 min, 70% B; 35 min, 5% B; 40 min, 5% B.

The effect of various olive leaf extract levels on the broilers’ growth performance

As shown in Table 3, compared with the control group, no significant change in ADG and ADFI of the broiler were observed until the supplementing level of OLE was more than 0.3% in the basal diet group, which was decreased from 28.18 to 23.43 g/day (p$=$0.001) as well as from 42.58 to 37.05 g/day (p$=$0.00037) in OLE0.4 group in the early period, respectively. Also, no significant changes in the FCR by all the OLE supplementation groups were observed (p$=0.443$) for the whole feeding period. However, a significant variation of FCR was indicated at the statistic mode of linear in the early and later period, respectively. In addition, compared with the control group without adding any OLE, the rate of mortality and culling for broilers tended to be decreased when the addition of OLE increased in the range from 0.1% to 0.3%. The lowest rate (1.11%) of mortality and culling emerged in the OLE0.3 group. However, the rate (12%) of mortality and culling for broilers obviously increased in the OLE0.5 group when the addition of OLE continued to increase.

Table 3. Effect of different supplementation levels of olive leaf extract on the growth performance in broilers.

Items of growth performance	Groups						SEM	p-Value
	Control	0.1% OLE	0.2% OLE	0.3% OLE	0.4% OLE	0.5% OLE		ANOVA
Initial body weight at 7 days, g	183.15 ± 0.13	184.21 ± 0.26	185.65 ± 0.35	186.47 ± 0.48	182.98 ± 0.36	184.08 ± 0.29	1.38	0.359
Early period (1 ∼ 21 days)
ADG, g/day	28.18 ± 1.08^a	28.13 ± 1.03^a	27.76 ± 0.56^ab	28.32 ± 0.78^a	23.43 ± 0.71^c	25.31 ± 1.01^bc	2.02	0.001
ADFI, g/day	42.58 ± 0.96^a	42.19 ± 1.07^a	42.30 ± 0.60^a	42.83 ± 0.67^a	37.05 ± 0.59^c	39.87 ± 0.63^b	2.27	0.00037
FCR, g/g	1.52 ± 0.03	1.50 ± 0.03	1.53 ± 0.02	1.52 ± 0.03	1.59 ± 0.04	1.58 ± 0.05	0.04	0.326
Later period, 22 ∼ 35 days
ADG, g/day	63.10 ± 1.54	65.05 ± 1.53	66.37 ± 1.77	66.13 ± 2.28	61.19 ± 1.11	64.75 ± 2.07	1.97	0.310
ADFI, g/day	122.77 ± 2.20^a	120.79 ± 1.93^a	125.64 ± 2.88^a	126.69 ± 3.78^a	111.03 ± 1.25^b	117.39 ± 4.67^ab	5.81	0.010
FCR, g/g	1.95 ± 0.03^a	1.86 ± 0.03^ab	1.89 ± 0.03^ab	1.92 ± 0.04^ab	1.82 ± 0.01^b	1.81 ± 0.05^b	0.06	0.063
The whole period, 1 ∼ 35 days
ADG, g/day	44.97 ± 1.10^ab	46.14 ± 0.98^a	46.68 ± 0.99^a	46.46 ± 1.25^a	42.26 ± 0.72^b	43.55 ± 1.18^ab	1.78	0.031
ADFI, g/day	84.42 ± 1.66^a	82.77 ± 1.55^a	84.71 ± 2.01^a	86.00 ± 2.45^a	74.04 ± 0.89^b	80.59 ± 3.58^a	4.36	<0.001
FCR, g/g	1.88 ± 0.05	1.80 ± 0.06	1.82 ± 0.03	1.85 ± 0.03	1.75 ± 0.02	1.86 ± 0.11	0.05	0.443
Death and culling rate, %	7.29 ± 1.02^b	5.21 ± 0.85^c	5.39 ± 0.79^c	1.11 ± 0.23^d	7.5 ± 1.08^b	12.08 ± 1.51^a	3.60	0.016

Note. ^a,bMeans within a row with no common superscripts differ significantly (p < 0.05); ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio. A varied ranging 0.1% OLE to 0.5% OLE indicate a different OLE level supplemented into daily diet of broiler.

The effect of various olive leaf extract levels on the meat quality and antioxidation of breast muscle meat in the broilers

As shown in Table 4, compared with the control group, the cooking losses in the OLE0.4 and OLE0.5 groups were increased from 13.00% to 20.41% as well as from 13.00% to 21.78% (p = 0.03), respectively. The brightness, yellowness and redness levels of the breast muscle were not significant for all the OLE groups. Table 4 also indicated that the shear forces ranging from OLE0.1 to OLE0.3 groups were decreased from 28.30 N to 18.87 N (p$=$0.03). As seen in Figure 2, the MDA level of the breast muscle meat dramatically decreased from 1.75 nmol/mgprot to 1.04 nmol/mgprot in the OLE0.3 group (p = 0.016). The T-SOD activity was increased by 50.4%, and the GSH level was increased by 76.64% in the OLE0.3 group (p = 0.0036). The GSH-Px activity was strikingly increased by 120% in the OLE0.3. Also, The T-AOC activity gave rise to a marked increase from the OLE0.3 group (p = 0.0062).

Figure 2. Antioxidation of the breast-muscle meat of the broiler chickens; malondialdehyde (MDA, nmol/mgprot), total antioxidant capacity (T-AOC, U/mg prot), total superoxide dismutase (T-SOD, U/mg prot), L-glutathione (GSH, μg/mgprot) and glutathione peroxidase (GSH-Px, U/mgprot) were detected in the tissue. Values with asterisk in the figure show a significant difference at p < 0.05 by one-way measures analysis of variance (ANOVA), compared with the control. For each description, every test was executed in triplicate (n = 3).

Table 4. Effect of different additions of the olive leaf extract on the meat quality of breast-muscle in chicken broilers.

Breast muscle meat quality	Groups						SEM	p-Value
	Control	0.1% OLE	0.2% OLE	0.3% OLE	0.4% OLE	0.5% OLE		ANOVA
Lightness (L*)	49.26 ± 1.17^a	45.71 ± 1.38^ab	45.31 ± 0.54^b	45.02 ± 1.47^b	44.71 ± 0.81^b	48.11 ± 1.42^ab	1.87	0.052
Redness (a*)	1.49 ± 0.22^b	2.28 ± 0.33^ab	2.67 ± 0.50^ab	3.06 ± 0.42^a	2.45 ± 0.39^ab	2.72 ± 0.39^ab	0.54	0.118
Yellowness (b*)	10.77 ± 0.53^a	8.37 ± 1.10^ab	9.33 ± 0.71^ab	8.98 ± 0.98^ab	7.58 ± 0.50^b	8.49 ± 0.77^ab	1.08	0.131
Shear force, N	28.30 ± 1.13^a	19.54 ± 1.13^b	20.54 ± 1.74^b	18.87 ± 3.13^b	23.66 ± 1.48^ab	26.53 ± 1.38^a	3.90	0.003
Cooking loss, %	13.00 ± 2.21^c	11.06 ± 0.79^c	10.02 ± 0.76^c	17.46 ± 3.65^b	20.41 ± 2.85^a	21.78 ± 2.10^a	4.97	0.003

Note: ^a,bMeans within a row with no common superscripts differ significantly (p < 0.05).

OLE: olive leaf extract.

The effect of various olive leaf extract levels on the biochemical index and antioxidation of broiler serum

As indicated in Table 5, compared with the control group, the urea nitrogen concentrations of the OLE0.1, OLE0.4 and OLE0.5 groups were increased by 158.53% (p = 0.0008), 141.46% (p = 0.0013) and 151.22% (p = 0.0008), respectively. OLE presented no significant effect on the serum of concentrations of total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and HDL/LDL in the broilers (p = 0.22), as seen in Figure 3. For antioxidant performances from Figure 4, in the case of OLE0.3, the OLE resulted in an increased level of T-SOD activity (p = 0.00013) and decreased levels of MDA in the broiler serum (p = 0.0029); the T-AOC and catalase (CAT) activities were considerably increased by 40.40% (p = 0.021) and 22.28% (p = 0.04), respectively. The total bilirubin concentrations of the OLE0.2 group were increased by 96.66% (p = 0.0009).

Figure 3. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) performances of the broiler chickens fed by olive leaf extract. Histograms with asterisk in the figure show significant difference at p < 0.05 by ANOVA, compared with the control. For each description, every test was executed in triplicate (n = 3).

Figure 4. Antioxidation of the blood serum of the broiler chickens; MDA (nmol/mg prot), T-SOD (U/mg prot)), T-AOC (U/mg prot) and catalase (CAT, U/mg prot) were detected. Values with asterisk in the figure indicate a significant difference at p < 0.05 by ANOVA, compared with the control. For each description, every test was executed in triplicate (n = 3).

Table 5. Effect of different additions of the olive leaf extract on the serum biochemical indexes in chicken broilers.

Serum biochemical indexes	Groups						SEM	p-Value
	Control	OLE0.1	OLE0.2	OLE0.3	OLE0.4	OLE0.5		ANOVA
Total protein, g/L	36.18 ± 2.99	40.12 ± 3.61	34.53 ± 1.62	34.08 ± 1.38	33.07 ± 1.65	35.39 ± 1.28	2.48	0.336
Albumin, g/L	13.87 ± 0.91	12.26 ± 1.33	14.41 ± 1.04	12.08 ± 1.10	13.81 ± 0.39	12.44 ± 0.73	1.00	0.392
Urea nitrogen, mmol/L	0.41 ± 0.05^b	1.06 ± 0.21^a	0.64 ± 0.07^b	0.54 ± 0.05^b	0.99 ± 0.09^a	1.03 ± 0.12^a	0.28	0.0008
Triglyceride, mmol/L	0.34 ± 0.05^b	0.52 ± 0.09^ab	0.49 ± 0.08^ab	0.54 ± 0.04^b	0.45 ± 0.03^ab	0.61 ± 0.09^a	0.09	0.111
Total cholesterol, mmol/L	4.77 ± 0.23	4.97 ± 0.39	4.75 ± 0.17	5.64 ± 0.31	5.64 ± 0.26	5.63 ± 0.28	0.45	0.044
Creatinine, μmol/L	427.10 ± 4.09	439.49 ± 7.95	422.85 ± 8.74	434.55 ± 1.77	429.32 ± 3.52	433.13 ± 3.21	5.89	0.014
Alkaline phosphatase, kings U/100 mL	161.97 ± 31.88^b	103.34 ± 18.71^d	182.90 ± 73.03^a	134.59 ± 25.15^c	93.51 ± 13.70^d	196.90 ± 53.56^a	42.22	0.170
Total bilirubin, μmol/L	12.89 ± 3.79^c	23.99 ± 2.37^ab	25.35 ± 2.24^a	17.19 ± 2.24^bc	17.87 ± 2.46^abc	22.86 ± 2.86^ab	4.81	0.359
Alanine transaminase, U/L	6.48 ± 1.68	7.10 ± 1.17	9.50 ± 1.09	6.69 ± 0.70	8.90 ± 0.44	7.70 ± 1.03	1.23	0.418
Aspartate aminotransferase, U/L	35.26 ± 5.39^ab	37.49 ± 3.48^ab	37.74 ± 2.58^ab	37.84 ± 3.08^ab	52.77 ± 12.95^a	32.22 ± 3.33^b	7.14	0.012

Note. ^a,bMeans within a row with no common superscripts differ significantly (p < 0.05). OLE0.1, OLE0.2, OLE0.3, OLE0.4 and OLE0.5 indicates varied OLE ranging from 0.1% OLE to 0.5% OLE supplemented into daily diet of broiler.

The effect of various olive leaf extract levels on the caeca microbiota in the broilers

As indicated in Figure 5, the caecal microbiota of the broilers presented different cases depending on the levels of the supplemented OLE concerning the intensity of total bacteria, E. coli, Lactobacillus and Bifidobacterium. The OLE0.3 group exhibited a decreased intensity of E. coli (p $=$ 0.004) and a noticeably increased intensity of Lactobacillus (p = 0.016) and Bifidobacterium (p = 0.038). Specifically, compared with the group, about 12% intensity of E. coli was decreased, and about 11% intensity of Lactobacillus and 13% intensity of Bifidobacterium were increased when the broilers were supplemented by 0.3% OLE. No significant variation in total quantities of bacteria was observed throughout the OLE groups.

Figure 5. Effects of different levels of olive leaf extract on the intensity of caeca-chyme microbiota in the broilers (log₁₀ copies/g).

Discussion

OLE, a well-recognised natural antioxidant, is widely applied in functional foods due to the abundance of oleuropein, flavonoids, phenolic acids and other phenols (Moudache et al. 2016). However, knowledge concerning OLE used as a broiler-chicken additive is still insufficient. Throughout the feeding period of 35 days, the addition of OLE such as 0.4% or 0.5% dramatically reduced the ADFI of broilers because the high dosage of OLE would seriously affect the palatability of the diet (Cecchi et al. 2013). Because of the bitter taste from oleuropein in the OLE supplemented into the basal diet, the feed intake might decrease and thereby give a lower body weight gain. Likewise, a similar situation happened in feed intakes as well. Moreover, no significant difference in the feed conversion ratio induced by OLE was observed. Especially, 0.3% OLE supplemented into the broilers’ basal diets could significantly decrease the death rate, as Table 2 indicated. This was possible ascribed to the appropriately higher oleuropein level (1.545 g/kg diet) and the other polyphenol level for synergism efficacy. However, excessive intake doses of OLE such as 0.4% OLE or 0.5% OLE obviously increased the death rate. Generally, ∼6% of mortality and culling for broilers could be produced by plant extract. In this study, the olive-leaf-extract itself has a bitter taste, especially for the high dose of supplementation such as 0.5% OLE, the intake of the dietary became greatly reduced, giving rise to a significant resistance to diseases and even death. Furthermore, the mortality rates in chickens could happen even under standard reared condition reaching ∼5% (Pakdel et al. 2002) and in whom ascites was induced reaching 23%. Because ascites syndrome, is a common and major problem in broiler poultry, especially in the case of poor-managed flocks, considerably deteriorates broiler performance (Luger et al. 2001).

Flesh colour is one of the most intuitive indexes to evaluate meat quality. In this work, the CIELAB evaluation system was used to comprehensively examine the changes in muscle colour. OLE obviously decreased the breast muscle brightness of broiler chickens when the supplemented OLE was over 0.2%. The haem content in the breast muscle is positively related to the redness level and is negatively related to the brightness (Sirri et al. 2009). After feeding OLE, the brightness of the broiler breast muscle notably decreased. These changes were responsible for the haem level in the muscle. Breast muscle maturation often occurs after broiler chickens are slaughtered along with autoxidation of partial haem pigments (Rhee and Ziprin 1987). OLE has an excellent free-radical-scavenging capacity and acts as a strong antioxidant. The antioxidation of OLE inhibited haem autoxidation, thereby reducing the brightness value of the breast muscle and improving the redness of the breast muscle. Shear force, another important parameter of muscle tenderness, has received much attention due to its popular chewing taste. The higher the shear force for the broiler muscle, the lower the tenderness (Dean et al. 1972). OLE ranging from 0.1% to 0.3% greatly decreased the shear force of the broiler breast muscle, suggesting that an appropriate level of OLE could enhance the taste of chicken breast muscle and make the muscle more delicate. This is because the oxidative stress of the broilers before slaughter, transportation, or feeding conditions gave a high level of reactive oxygen radicals that induced a low quality of the breast muscle (Lund et al. 2011). OLE extensively improves oxidative stress as a strong radical scavenger due to a very high level of oleuropein and polyphenol.

GSH, a tripeptide nonprotein sulfhydryl compound composed of glutamate, cysteine and glycine, is a pivotal small-molecule antioxidant in cells. GSH is capable of eliminating peroxidation products and various heterogenics of broiler chickens in vivo to regulate the cell cycle and maintain the reducing power of intracellular sulfhydryl groups (Oetari et al. 1996). In addition, SOD is reckoned as the first antioxidant defence against free radical attacks that can result in a catalytic endogenous superoxide anion through a disproportionate reaction into hydrogen peroxide. GSH is catalysed by GSH-Px with H₂O₂ to transfer lipid peroxide into water and fatty alcohols only to remove or lower the oxidative toxicity induced by oxygen-free radicals. In this study, 0.3% supplement of olive leaf extract supplementation significantly increased GSH levels, GSH-Px and T-SOD activity in breast muscle. On one hand, MDA, one of the metabolic products of lipid peroxides and closely related to GSH-Px, was drastically reduced in broiler serum. The decreased MDA level and the remarkably increased GSH-Px activity of the breast meat showed that OLE enables the inhibition of oxidation and prolongs its shelf life. This is because OLE possesses great antioxidant potency in diminishing reactive oxygen species and lipid peroxidation. These findings were in good line with the previous observation demonstrated in another work (Senturk and Yildiz 2018), reflecting a noteworthy improvement in the health and oxidative situation of broiler chickens. This finding was consistent with the results that suggested that the appropriate addition level of OLE was beneficial for breast muscle quality. On the other hand, the CAT activity acting as H₂O₂ resistance was markedly increased and enhanced the antioxidant performance (Osman et al. 2020). Therefore, the increased antioxidant activity of OLE on the breast muscle of the broilers possibly resulted from these joint efforts, such as the synergistic effectiveness of bioactive polyphenols.

Total bilirubin is the sum of direct bilirubin and indirect bilirubin. The results showed that a 0.2% addition of OLE into the broiler chicken radically increased the level of total bilirubin in the serum, whereas addition of 0.3% or 0.4% of OLEs showed a mild increasing trend. The increased level of total bilirubin is possibly related to other causes. For instance, the relatively low concentrations of bilirubin in plasma can effectively prevent the oxidation of fatty acids from being bound to albumin as well as eliminate free radicals induced in vivo (Wu et al. 1996). Additionally, decreased LDL and increased HDL levels were observed (Bahşi̇ et al. 2016). Prior studies demonstrated that oleuropein can protect low-density lipoprotein against oxidation, thereby lowering LDL (bad cholesterol) levels. OLE presents extremely high antioxidative attributes because of this reason, at least in part. This behaviour is available to the human biosystem whereas it did not produce protection of LDL in broilers in this study, possibly owing to a significant discrepancy in the digestion system and metabolism pathway between them (Polzonetti et al. 2004). The alanine aminotransferase and alkaline phosphatase activities in the serum were obviously increased in addition to hepatic fibrosis when OLE concentrations were supplemented over 0.5% compared with the control (Arantes-Rodrigues et al. 2011).

For microbial regulation of OLE in the broiler caeca, the high dose of oleuropein and polyphenol enrichment in OLE gave a significant quantitative decrease in terms of E. coli and a notable increase in terms of Lactobacillus and Bifidobacterium quantity. All of these factors can largely improve the broiler’s gut health, which is thereby very helpful in promoting meat quality (Chen et al. 2019; Dev et al. 2020). Specifically, E. coli is distributed among poultry of all ages and is a natural inhabitant of the gut in poultry. Escherichia coli is commonly referred to as colibacillosis. It could lead to woody breast meat. The woody breast myopathy of broilers was affected by broiler strain and diet (Zhang et al. 2018). Reducing the abundance of Escherichia and Shigella in the broiler caeca (Eeckhaut et al. 2016) has potential in improving broilers’ gut health and reducing woody breast development (Zhang et al. 2021a). Also, some cell surface proteins from Lactobacillus could immobilise E. coli and prohibit its propagation (Tsou et al. 2017). Secoiridoids, flavonoids and simple phenols in OLE that inhibit E. coli resulted from alterations in cytoplasmic membranes and the cell wall (Rodríguez et al. 2009), and the E. coli abundance was dramatically decreased by the plant extracts. However, the additive/synergic effects of the different products that are present in this extract can also play a key role, making it difficult to discriminate and correlate each activity to specific compounds (Vezza et al. 2019). For instance, previous observations demonstrated that OLE had an antioxidant activity significantly higher than vitamin C and E, due to the synergy between flavonoids, oleuropeosides and substituted phenols (Benavente-Garcı́a et al. 2000). Also, a combination of quercetin and caffeic acid for each individual compound at 5 μm in a 1:1 mixture indicated an additive effect of antioxidation (Meyer et al. 1998). A combination of rutin and α-tocopherol for each individual compound at 0.1 ∼ 1.0 mM in 1:1 ratio indicated a synergism effect of antioxidation in vitro (Kancheva et al. 2007; Kancheva 2009). The gut microbiota functions in gut health and nutrition in poultry industry. Probiotics and prebiotics are generally utilised for modulating gut microbiota. In this scenario, oleuropein and hydroxytyrosol enable adjusting the composition of gut microbiota and improving gut integrity (Sarica and Urkmez 2016). One possible reason for inhibiting the growth of E. coli is that polyphenols can bind to bacterial cell membranes to disturb membrane function, for example, altering the permeability and proton motive force through the loss of H⁺-ATPase and membrane-related functions (Lin et al. 2005; Cardona et al. 2013). A possible description for the resulting effect of phenols on bacterial growth such as Lactobacillus and Bifidobacterium is that they enable using these phenols as substrates. Lactobacilli can metabolise the polyphenols in the growing process and polyphenols provide energy for cells in turn. Besides, these polyphenols can intensify nutrient consumption such as carbohydrates (García-Ruiz et al. 2008). In a word, polyphenol supplementation in poultry can modulate caecal microbiota species and levels, as exemplified in our study.

Conclusions

In this study, we observed that the OLE containing a high amount of oleuropein, polyphenols and flavonoids, can be effectively applied as an alternative additive of synthetic antioxidant for improving meat quality, increasing antioxidation and caecal health. Also, 0.3% OLE supplemented into the basal diet of broiler chickens gave rise to superior multiple performances. Specifically, the meat quality and antioxidation of broilers were greatly improved: including a considerate reduction in shear force of the meat, a notable enhancement in the levels of the antioxidant enzymes (T-AOC, T-SOD, GSH-Px and CAT) and GSH, as well as a decrease in the MDA level. Moreover, OLE plays a key role in diminishing the intensity of E. coli and enlarging the intensity of Lactobacillus and Bifidobacterium in terms of caecal microbiota. Overall, a proportional addition of OLE into the basal diet is potentially used as an effective natural antioxidant and a caecum-health promoter for enhancing meat quality in broilers.

Ethical approval

The experiment was approved by the Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China, NAU-2018320122000011).

Acknowledgements

The authors thank Prof. Wang Tian for providing great support towards broiler management and Dr. Zhang Hao for caecal microbiota analysis from the College of Animal Science and Technology at Nanjing Agricultural University.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Natural Science Foundation of China [31800614] and Fundamental Research Funds of CAF [CAFYBB2017ZD004].