Effect of Different Combinations of Methanolic Extract of Moringa oleifera and Thymus vulgaris on Production Performance, Gut Morphology, Hematology and Nutrient Digestibility in Broilers

Majid Ali1*, Naila Chand1, Sarzamin Khan1, Shakoor Ahmad2 and Muhammad Tahir3

1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan

2College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan

3Department of Animal Nutrition, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan

ABSTRACT

The present study was designed to find the effect of methanolic extract of Moringa oleifera and Thymus vulgaris on the performance, hematology, digestibility and gut health in broiler chickens. A day-old 270 broiler chicks (Ross-308) were randomly assigned to 6 treatment groups with 3 replicates and 15 birds per replicate. CON (Control) group; M100 (M. oleifera 200mg/L); T100 (T. vulgaris 300mg/L); M50T50 (M. oleifera 100mg/L and T. vulgaris 150mg/L); M75T25 (M. oleifera 150mg/L and T. vulgaris 75mg/L); and M25T75 group (M. oleifera 50mg/L and T. vulgaris 225mg/L). Methanolic extract of both M. oleifera and T. vulgaris were supplemented in drinking water. The duration of the experiment was 42 days. The results showed significantly higher feed intake, body weight gain, feed conversion ratio, broiler performance efficiency factor, and broiler farm economy index in the M50T50 diet group. Digestibility of ash, dry matter, ether extract, crude fiber, and nitrogen-free extract were not affected except for crude protein which was significantly higher in the M50T50 diet group. Similarly, hematological parameters were not affected, while villus height, width, and crypt depth were significantly improved in the M50T50 diet group. These findings demonstrate that the supplementation of methanolic extract of M. oleifera and T. vulgaris in drinking water either alone or in combination improves the production performance, nutrient digestibility and gut health in broilers.

Article Information

Received 18 December 2022

Revised 20 February 2023

Accepted 10 March 2023

Available online 05 May 2023

(early access)

Published 10 May 2024

Authors’ Contribution

MA study design, Animal trial, laboratory experiment, statistical analysis, and writing. NC study design, feed formulation, data evaluation, manuscript review. SK, SA and MT data analysis, data evaluation, manuscript review.

Key words

Moringa oleifera, Thymus vulgaris, Gut health, Digestibility, Production performance

DOI: https://dx.doi.org/10.17582/journal.pjz/20221218121209

* Corresponding author: majidaup1@gmail.com

0030-9923/2024/0003-1477 $ 9.00/0

Copyright 2024 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The poultry industry is the second largest sector of Pakistan and for the last several decades performed an emerging role in the country’s economic sector contributing 5.77% to the agriculture sector, 26.7% to total meat in the form of superior protein, and 1.3% to national GDP (PPA, 2018-2019). More than 1.5 million people inthe country are involved in the poultry industry in the form of employment. In the last few decades, different synthetic and chemical antibiotics were used as growth promoters that harm the natural physiology of the bird microflora and also induced drug resistance in humans and bacteria. There is an intense need to decrease the application of synthetic drugs such as antibiotics in poultry production because of antimicrobial resistance (Borazjanizadeh et al., 2011). This is possible through the inclusion and use of phytogenic plants which replace antibiotics and work as growth promoters in poultry production. Therefore, poultry scientists are involved to investigate and use unconventional plants and plant by-products to decline the heavy economic damage in the form of different microbial diseases and improve poultry health and production. To date incorporation of phytogenic extract generated an essential role in the improvement of the poultry industry in the form of health and performance. Moringa oleifera is a drought, a resistant and fast-growing tree grown in sub-Himalayan zones of northern Pakistan, Afghanistan, India, and Bangladesh. It is growing worldwide now in the subtropics and tropics zone (Fahey et al., 2001). Siddhuraju and Becker (2003) reported that M. oleifera contains active ingredients such as crude protein 27.51%, ether extract 22.3%, crude fiber 19.25%, ash 7.13%, and dry matter 76.53%. It has also been reported to contain caffeic acid and chlorogenic acid also which are known for antioxidant effects. M. oleifera reduces and inhibits the degradation of amino acids and positively improves the growth performance of birds (Alagawany et al., 2017). Ebenebe et al. (2012) have reported that M. oleifera supplementation improves the feed conversion ratio of meat-type birds. Supplementation of M. oleifera in the feed of broiler and layer birds suppresses the gut microbial count such as C. perfringens, coliforms, and E. coli, and also has a gut integrity role (Arif et al., 2019). Thymus vulgaris known for cosmetic, medical, and culinary purposes. It has strong growth stimulant properties mostly used as an alternative for synthetic and chemical materials in the poultry industry. T. vulgaris have some important functions such as antioxidant, antiseptic, antispasmodic, and expectorant (Hertrampf, 2001; Adu-Darwish et al., 2009). T. vulgaris contains thymol which is the fundamental active ingredient and also contains 21-56% volatile oils, carvacrol (5-isopropyl-2-methyl phenol), and phenolic compound (5-methyl-1-2-isopropyl phenol) with well-known antimicrobial activity (Rahimi et al., 2011). Thyme contains intrinsic bioactivities and also has appetite and digestion-stimulating properties (Herendez et al., 2004). This appetizing property increases feed utilization and growth performance (Bolukbasi et al., 2006; Al-Mashadani et al., 2011). Plant extract protects the absorptive surface and villi of the gut and intestine against toxins produced by pathogens mostly viruses and bacteria (Rolfe et al., 2000). Thyme in combination with other phytogenic significantly improves gut integrity by enhancing the villus height and width (Demir et al., 2003). To date, very less studies reported on the extract of M. oleifera and T. vulgaris but no such study reported on the combined use of M. oleifera and T. vulgaris extract on poultry health and performance. Therefore; the current study was designed to determine the effect of different combinations of methanolic extract of M. oleifera and T. vulgaris on overall performance, gut morphology, hematology, and nutrient digestibility in broilers.

MATERIALS AND METHODS

Preparation of methanolic extract of Moringa oleifera and Thymus vulgaris

M. oleifera and T. vulgaris fresh leaves were harvested and collected from local areas of Peshawar, Khyber Pakhtunkhwa Pakistan, and were separately air and shade dried because directly expose to extreme sun light effect the active ingredient of the plant leaves. For inhibiting the fungal growth, leaves were turned over at constant intervals for at least one week. After complete drying, the leaves were ground and sieved with a 0.15-mm sieve and made a fine powder. About 100 ml of absolute methanol and 200 gm powder of both M. oleifera and T. vulgaris leaves dissolved separately in soxhlet apparatus overnight. After 24 h about 50ml extract was dissolved in dimethyl sulfoxide and kept for 72 h. After 72 h a cohesive mass was obtained which contained 10 mg/ml of methanolic extract of M. oleifera and T. vulgeris approximately. Experimental diets are presented in Table I.

 

Table I. Composition of feed during the starter and finisher phase of experimental birds.

Ingredients (%)	Starter phase	Finisher phase
Corn gluten meal	1.98	7.14
Corn	54.1	60.2
Corn oil	2.18	2.70
Soyabeen meal	37.6	24.0
Limestone	0.81	0.67
Dicalcium phosphate	2.35	2.10
VM mixa	0.50	0.50
Salt	0.50	0.55
Lysine HCL	0.21	0.40
DL-Methionine	0.22	0.12
Cholin chloride	0.06	0.06
Threonine	0.12	0.10
Chemical composition
Crude protein (%)	23.0	21.0
ME (Kcal/kg)	3000	3150
Methionine (%)	0.54	0.44
Sulphur amino acid (%)	0.94	0.78
Lysine (%)	1.40	1.22
Calcium (%)	1.06	0.90
Phosphorus (%)	0.50	0.46
Threonine (%)	0.95	0.88

 

aVitamin-minral premix contains the following ingredient per kg of feed: vitamen A, (2,400,000 IU); vitamen E, (16,000 IU); vitamin K, 800 mg; vitamin D, 1,000,000 IU; vitamin B12, 6 mg; vitamin B6, 1000 mg; vitamin B2, 1600 mg; vitamin B1, 600 mg; folic acid, 400 mg; biotin 40 mg; niacin, 8000 mg; pantothenic acid, 3000 mg; antioxidant, 3000 mg; copper, 2000 mg; selenium, 60 mg; cobalt, manganese, 18,000 mg; 80 mg; iron, 1200 mg; iodine, 400; zinc and14,000 mg.

 

Experimental design and bird’s husbandry

A total of 270 days old chicks (Ross-308) were randomly assigned to six different treatment groups in a completely randomized design (CRD). Each treatment group was further divided into 3 replicates with 15 birds per replicate. The same basal diet was fed to birds of all experimental groups. Control group with (no supplementation); M100 (Supplemented with Moringa 200mg/L); T100 (supplemented with Thyme 300mg/L); M50T50 (supplemented with M. oleifera 100mg/L and Thymus vulgaris 150mg/L); M75T25 (supplemented with Moringa oleifera 150mg/L and T. vulgaris 75mg/L); M25T75 (supplemented with M. oleifera 50mg/L and T. vulgaris 225mg/L). All the supplementation was given in the drinking water. The optimum number of the drinker, feeder, and other requirements were provided to the birds in such a way that the bird’s genetics show the best potential results of all the experimental parameters. The experiment was extended for 42 days including 7 days of adaptation.

Performance parameters

Feed was offered to the broiler chicks ad libitum and daily feed intake was measured by subtracting the refused feed from the feed offered while weight gain was calculated by subtracting the initial body weight from the final body weight. The feed conversion ratio was calculated by dividing feed intake by weight gain x 100. Mortality was recorded throughout the experiment. The percent livability was calculated at the end of the experiment by the formula:

Livability (%) = Birds sold ÷ number of birds present at the beginning x 100

The broiler performance efficiency factor (BPEF) was computed by the formula;

BPEF= Bird live body weight in Kg ÷ feed conversion ratio x 100

while the broiler performance efficiency index was calculated by the formula:

BPEI= live weight in kg x % livability ÷ feed conversion ratio x growing period in days

Hematology and gut morphometry

Blood parameters were determined on the last day of the experiment and 3 to 5 ml of blood was collected from the wing vein of 03 birds of each replicate. Hemocytometer was used for the manual calculation of total white blood cells (WBCs) and total red blood cells (RBCs) (Campbell, 1995). The packed cell volume (PCV) was measured by a standard manual technique using microhematocrit capillary tubes and blood was centrifuged at 2500 rpm for 5 min. The cyanmethemoglobin methodology was implemented for the estimation of hemoglobin (HB) concentration in the blood. For gut morphometry, the procedure used by Shuaib et al. (2022) was followed.

Nutrient digestibility

Nutrient digestibility was calculated by using the indigestible and insoluble marker Cr2O3 on day 35. From each replicate, 5 birds were randomly shifted to metabolic cages on day 35 of the experiment for total excreta collection. The birds remained for 4 days in these metabolic cages provided with a ration containing 0.2% Cr2O3 as an indigestible marker. Fresh fecal samples were collected twice a day in a plastic bag and stored at -20°C. Excreta samples, ileal digesta, and ground feed were properly dried, ground, and sieved through a 0.5-01 mm sieve and kept at -20°C. Proximate analyses of feed and excreta samples were done as defined by (Adejumo et al., 2005). The concentrations of chromium were determined with the help of a UV absorption spectrophotometer (Shimadzu, Kyoto, Japan Shimadzu, UV-1201) as described by Williams et al. (1962). The nutrient digestibility was determined according to the procedure used by (Stefanello et al., 2020).

Statistical analysis

Complete randomized design (CRD) was used for the analysis and computing of the collected data. For significance, the difference means were reviewed by least significant difference (LSD) (Guide, 2010).

RESULTS

Results regarding feed intake, weight gain, feed conversion ratio, production traits, and gut health are shown in Table II. During the starter phase higher (P<0.05) feed intake was recorded in the M50T50 diet group but at the finisher phase in the M100, M50T50, and M75T25 diet groups. Overall, significantly higher feed intake was recorded in the M50T50 and M75T25 diet groups. Weight gain during the starter and finisher phases as well as overall was calculated higher (P<0.05) in the M50T50 diet group. At the starter, and finisher phases and an overall significantly better FCR was recorded in the M50T50 and M75T25 diet groups. The highest significant BPEF was noted for M50T50, M25T75, and M75T25 diet groups, and the highest broiler farm economy index was noted for the M50T50 group. Mortality was recorded as non-significant among the groups. The villus height and villus width were recorded higher (P<0.05) in the M50T50 diet group while the crypt depth had a higher (P<0.05) value in the control group. Results regarding nutrient digestibility and hematology are presented in Table III. Digestibility of dry matter, ash, crude fiber, ether extract, and the nitrogen-free extract was not affected but crude protein had a significantly higher digestibility in the M100, M50T50, and M75T25 diet groups than the all-other groups. Blood parameters were not affected (P<0.05) among the groups.

 

Table II. Effect of methonolic extract of Moringa oleifera and Thymus vulgaris alone or in different combinations on feed intake, weight gain, FCR, production traits and gut health of broiler birds.

Parameters	Groups						P value
	Control	M100	T100	M50T50	M75T25	M25T75
Feed intake (g)
Starter phase	982.3±7.35 c	1009±7.26 b	976.6±2.33 c	1048.3±3.28 a	1014.2±5.02 b	982±9.50 c	0.001
Finisher phase	2442.7±5.81 c	2745.3±11.7 a	2641.7±1.45 b	2780.7±3.93 a	2781.3±3.92 a	2668±23.3 b	0.003
Overall (day1-42)	3425±3.51 d	3754.7±5.36 b	3618.3±1.45 c	3829±38.6 a	3795.5±8.65ab	3650±15.8 c	0.002
Weight gain (g)
Starter phase	601.8±4.37 d	633.5±8.25 c	602.2±3.05 d	678.5±5.81 a	649.8±1.33 b	608.8±3.45 d	0.001
Finisher phase	1017±29.1 d	1399.3±12.3 c	1355.3±37.9 c	1650.7±10.7 a	1564.7±10.4 b	1342.3±8.25 c	0.002
Overall (day1-42)	1618.9±26.9 e	2032.9±18.6 c	1957.5±38.5 d	2329.2±9.07 a	2214.5±11.4 b	1951.2±11.7 d	0.003
FCR
Starter phase	1.63±0.01 a	1.59±0.01 b	1.62±4.60 ab	1.54±9.30 c	1.56±7.30 c	1.61±8.50 ab	0.004
Finisher phase	2.40±0.06 a	1.96±0.02 b	1.94±0.05 b	1.68±0.02 c	1.77±0.01 c	1.98±0.02 b	0.001
Overall (day1-42)	2.11±0.04 a	1.84±0.01 b	1.84±0.03 b	1.64±0.01 c	1.71±0.01 c	1.87±0.01 b	0.002
Livability (%)	93.7±2.22	100.0±0.00	100.0±0.00	100.0±0.00	100.0±0.00	97.7±2.22	0.366
BPEF (%)	82.3± 4.08 d	109.3± 1.21 b	95.2± 2.66 c	121.4±1.31 a	116.4±0.41 a	118.3±1.50 a	0.003
BFEI	1.95± 0.30 c	2.63± 0.32 b	1.96 ±0.17 c	3.32± 0.11 a	2.67± 0.22 b	2.72± 0.33 b	0.000
Mortality (%)	1.00±0.57	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.33±0.33	0.366
Gut health
Villus height(µm)	610± 4.08 c	824± 1.21 b	755± 2.66 bc	862±1.31 a	803±0.41 b	788±1.50 bc	0.003
Villus width (µm)	54± 0.30 c	68± 0.32 b	57±0.17 bc	74± 0.11 a	64± 0.22 b	59± 0.33 b	0.045
Crypt depth (µm)	68±1.23 a	59±0.53 b	56±2.78 b	47±2.22 d	56±2.78 b	53±2.22 c	0.003

 

Means with different superscripts in the same row to each treatment are significantly different at α= 0.05. Control, untreated; M100, supplemented with 100% Moringa oleifera; T100, supplemented with 100% Thymus vulgaris; M50T50, supplemented with 50% M. oleifera and 50% T. vulgaris; M75T25, supplemented with 75% M. oleifera and 25% T. vulgaris; M25T75, supplemented with 25% M. oleifera and 75% T. vulgaris; BFEI, broiler farm economy index; BPEF, broiler performance efficiency factor (%).

 

Table III. Effect of methonolic extract of Moringa oleifera and Thymus vulgaris alone or in different combinations on nutrient digestibility and hematology of broilers.

Parameters	Groups						P. value
	Control	M100	T100	M50T50	M75T25	M25T75
DM (%)	66.3±0.76	70.2±0.47	68.5±0.38	71.7±0.37	70.6±0.34	69.1±0.60	0.140
Ash (%)	41.5±3.53	45.6±2.44	43.8±0.11	47.7±0.53	44.9±1.00	44.7±0.40	0.348
CP (%)	70.3±0.25c	72.2±0.13ab	70.7±0.22c	73.0±0.16a	71.9±0.04ab	71.2±0.80bc	0.002
EE (%)	77.9±2.83	81.3±1.23	78.6±2.29	85.0±1.21	81.7±1.26	80.1±0.68	0.128
CF (%)	79.8±1.33	83.5±0.92	81.6±0.74	83.9±0.01	83.6±0.33	81.2±0.28	0.299
NFE (%)	80.4±0.95	83.5±1.39	80.8±1.15	85.5±0.93	84.7±0.46	80.4±2.31	0.053
RBC (1012/L)	1.98±0.03	2.30±0.06	2.14±0.05	2.50±0.07	2.38±0.30	2.08±5.35	0.122
WBC (109/L)	10.5±0.85	7.97±1.19	8.85±2.38	6.98±0.03	6.32±0.57	6.97±0.05	0.188
PCV (%)	28.2±2.43	30.4±1.00	30.6±1.00	32.5±1.70	31.0±0.86	30.0±2.08	0.632
HB (g/dl)	10.2±1.00	10.9±0.60	10.8±0.20	12.6±0.40	12.0±0.87	11.2±1.08	0.357

 

Means with different superscripts in the same row to each treatment are significantly different at α=0.05. DM, Dry matter; CP, Crude protein; EE, Ether extract; CF, Crude fiber; NFE, Nitrogen free extract; RBC, Red blood cells; WBC, White blood cells; PCV, Packed cell volume; HB, Hemoglobin.

 

DISCUSSION

In the current study, the M50T50 diet group exhibit the highest performance in the form of feed intake, body weight, and improved FCR while all the performance parameter were observed better in the phytogenic (M. oleifera and T. vulgaris) combination group than in the control group. Our results are similar to the findings of Feizi et al. (2013) who reported that thyme extracts contain thymol which activates broiler’s digestive system by secreting endogen a digestive enzyme that speeds up the absorption rate of the intestine and subsequently improved the utilization and absorption of feed. Our results are also in line with the results of Mikhail et al. (2020) who stated that kaempferol and quercetin flavoring glycosides are abundant in the M. oleifera leaf. Provision of M50T50 significantly increased the bird’s final body weight gain which might be due to synergetic effect of both M. oleifera and T. vulgeris. M. oleifera contains flavonoids having antimicrobial activity and the thymus contains thymol and carvacrol principle active molecules which increased the palatability of feed and also help to boost the nutrient absorption of broilers which ultimately leads to increased weight gain. Similarly, Alabi et al. (2017) reported that the phenolic compounds containing flavonoid content present in M. oleifera seeds had an encouraging effect on broiler growth rate. Alagawany et al. (2017) reported that M. oleifera could decrease and inhibit the degradation of amino acids and hence positively improve growth performance. Similarly, Feizi et al. (2013) supplemented the birds with T. vulgaris (200cc/1000 liter) and reported higher body weight gain, and Saki et al. (2014) mixed 0.2 ml/liter extract of T. vulgaris in drinking water and reported higher weight gain. Similarly, the best FCR was recorded in the M50T50 diet group which might be due to the synergetic effect of both M. oleifera and T. vulgaris. Similar results were also reported by Kout et al. (2015), who fed MOLM to birds with 0.2% concentration in the feed. Ebenebe et al. (2012) also found better FCR by supplementation of M. oleifera. Our results are also in line with Ocak et al. (2008) by the inclusion of 2% thyme. Cross et al. (2003), Dahal and Farran (2011) also favor our results. Supplementation of the methanolic extract significantly improved the broiler performance and economical parameters which are similar to the findings of Zanu et al. (2012) who reported improved feed cost in meat-type birds by supplementation of M. oleifera mix with fish meal. Abbas et al. (2012) also concluded that 10% Moringa leaf meal is cost-effective in broilers. Supplementation of methanolic extract of M. oleifera and T. vulgaris significantly (P˂0.05) affected the crude protein which is in line with the results of El-Badawi et al. (2014) who fed 0.15 or 0.30% M. oleifera dry leaves powder to the rabbits in the ration. Supplementation of methanolic extract of M. oleifera and T. vulgaris significantly (P˂0.05) increased the height and width of the ilium portion of villi in all supplemented groups which is due to the synergetic effect of both phytogenic methanolic extract and this synergetic effect increases the surface area which directly increases the absorptive capacity and ultimately leads to increased digestibility and nutrient utilization. Generally, crypt depth decreased by increasing villus height and width and similarly, our findings also exhibited a higher decrease in crypt depth in group M50T50. Similarly, Saeed et al. (2018) found that broilers fed with herbs significantly stimulate the jejunum histology which enhanced the absorption of nutrients and growth performance.

CONCLUSION

The administration of methanolic extract of M. oleifea and T. vulgaris enhanced the utilization of feed efficiency, and body performance and also potentially modulate and improve gut health which beneficially improves the crude protein digestibility. Hence supplementation of methanolic extract of M. oleifera and T. vulgaris alone and in combination form enhanced the broilers’ performance but the most effective improvement was exhibited by the M50T50 group in drinking water on the production performance, nutrient digestibility, and gut health in broilers.

ACKNOWLEDGMENT

We acknowledge the staff of the Department of Poultry Science and Faculty of Animal Husbandry and Veterinary Sciences (FAHVS), The University of Agriculture Peshawar, Pakistan who provided technical and laboratory facilities.

Funding

No funding was provided /received for this study.

IRB approval

The experimental work was approved by the Advanced Studies and Research Board (ASRB) (No.712/ASRB-56/UAP), The University of Agriculture, Peshawar, KP, Pakistan.

Ethical statement

This study was approved by the animal welfare and care committee of the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan, and all the measures and tools was considered to minimize the pain and discomfort of birds during the conduction of this experiment.

Statement of conflict of interest

The authors have declared no conflict of interest.

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