Effect of diet supplemented with phytogenics and protease enzyme on performance, serum biochemistry and muscle histomorphology in broilers

ABSTRACT

Phytogenics are being considered a suitable alternative to antibiotics as growth promoters. In the current experiment, five hundred 40-day-old Cobb 500 broiler chickens were randomly allotted to five treatments as follow: control (basal diet only), basal diet + Ajwan powder (10 mg/kg of feed), basal diet + Coriander powder (10 mg/kg of feed), basal diet + black Cumin powder (10 mg/kg of feed), basal diet + protease enzyme (30,000 IU/kg). The results showed that feed intake was significantly (P < 0.05) higher in Coriander and protease treated birds at the end of the starter and finisher phases. On overall basis, body weight was significantly (P < 0.05) higher in protease, black Cumin and Coriander treated birds compared to the control. Feed conversion ratio was significantly (P < 0.05) improved in black Cumin and protease enzyme treated birds compared to the control. The results indicated that muscle fibre diameter, muscle fibre cross sectional area, muscle fascial diameter and muscle fascial cross sectional area were significantly (P < 0.05) higher in protease and black Cumin supplemented birds compared to the control. From the results of the present study, it was concluded that the performance and muscle histology were significantly improved in protease enzyme and phytogenics treated broiler.

KEYWORDS:

• Broiler
• black cumin
• protease
• performance
• muscle histology

Introduction

Antibiotic under controlled doses have played a key role in poultry production (Abudabos et al. 2017; Hafeez et al. 2020). However, these antibiotics are under serious criticism in many countries of the world due to their residues in meat and antimicrobial resistance against the bacteria (Khan et al. 2012a; Ahmad et al. 2020; Ali et al. 2019). Therefore, recently the non-antibiotic substances have been given immense importance (Khan et al. 2012b; Abudabos et al. 2016; Akhtar et al. 2020). Herbs and spices are also added into the poultry diet to improve feed intake and weight gain (Alzawqari et al. 2016; Chand et al. 2018; Safiullah et al. 2019; Wahab et al. 2019).

Coriander (Coriandrum sativum) is known as both a spice and an herb. This has been used as remedy in traditional medicine. In literature, it has been reported for hypocholesterolemic, hypolipidemic, antioxidant, and antifungal activities (Hosseinzadeh et al. 2014). The essential oil of coriander consisted of geraniol, camphor, geranyl acetate, terpinene, pinene and linalool (Burdock and Carabin 2009). Ajwan (Carum copticum) is an herbaceous plant which has stimulatory effect on digestion (Valiollahi et al. 2014). It can also be used against fungi and bacteria and can act as an antioxidant (Falaki et al. 2016). Black cumin (Bunium persicum) is another potential plant which can be utilized for the purpose of treatment due to antifungal properties (Sekine et al. 2007). In ancient times, the seeds of black Cumin were being utilized in the Persian medicine to formulate a carminative mixture and for the treatment of diarrhoea (Tehseen et al. 2016). Seeds of black Cumin can be used for the treatment of dyspepsia and diarrhoea. Other properties include appetizer, antimicrobial activity and antioxidant (Sacchetti et al. 2005).

Protease is an enzyme, which is responsible for proteolysis. Exogenous protease supplementation has been reported for improved crude protein digestibility in broilers (Freitas et al. 2011). In addition, studies on exogenous supplementation of protease enzyme improved the digestibility in broiler chicks and turkeys (Cowieson et al. 2017). Other studies showed no effects (Erdaw et al. 2017) or reduced effects (Walk et al. 2018) in response to exogenous protease supplementation. Most of the studies in the published literature have either used single plant or multiple plants in comparison with control, however, we did not find any study which compared plants with protease enzyme. Therefore, the objective of the present study was to compare different herbs with protease enzyme on the performance, muscle histology and serum biochemistry of broiler.

Materials and methods

This study was approved by the ethical committee on animal welfare and rights approved by the Faculty of Animal Husbandry & Veterinary Sciences, The University of Agriculture, Peshawar, Pakistan.

Birds and experimental design

The experiment was approved under the protocol approved by the Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture, Peshawar. Five hundred 40-day-old Cobb 500 broiler chickens were randomly allotted to five treatments and five replicates as follow:

1. Control (basal diet only)

2. Basal diet + Ajwan powder (10 mg/kg of feed)

3. Basal diet + Coriander powder (10 mg/kg of feed)

4. Basal diet + black cumin powder (10 mg/kg of feed)

5. Basal diet + protease enzyme (30,000 IU/kg)

Birds were fed a starter (1–21 days) and finisher diet (22–42 days) that meet the recommendation of NRC (1994) as shown in Table 1. Ajwan, Coriander, black Cumin and protease enzyme were mixed with the basal diet according to the experimental design. The birds were housed in cages with a density of 9.5 birds/m². Birds were reared on concrete floor with wood shaving as bedding material. Temperature was initially adjusted at 35 ± 2°C and then gradually reduced and maintained at 25 ± 2°C. Relative humidity was about 65%. The chickens were vaccinated against the diseases according to the standard protocol.

Table 1. Composition of basal feed and chemical composition.

Ingredients	Starter phase	Finisher phase
Corn	55.5	56.00
Soybean meal	27.4	26.5
Canola meal	6.5	6.00
Sunflower meal	3.2	3.5
Vegetable oil	1.8	1.9
Molasses	1.00	1.00
Dialcium phosphate	1.9	1.9
Limestone	1.00	1.00
NaCl	1.00	1.00
NAHCO3	0.2	0.2
Vitamin mineral premix	0.3	0.3
Chemical composition
ME, kcal/kg	3000	3150
Crude protein, %	23.5	21.30
Methionine, %	0.55	0.44
Lysine, %	1.42	1.23
Sulfur amino acids, %	0.96	0.80
Threonine, %	0.95	0.85
Calcium, %	1.05	0.90
Phosphorus, %	0.50	0.45

Note: Vitamin-mineral premix contains in the following per kg: vitamin A, 2,400,000 IU; vitamin D, 1,000,000 IU; vitamin E, 16,000 IU; vitamin K, 800 mg; vitamin B1, 600 mg; vitamin B₂, 1600 mg; vitamin B₆, 1000 mg; vitamin B₁₂, 6 mg; niacin, 8000 mg; folic acid, 400 mg; pantothenic acid, 3000 mg; biotin 40 mg; antioxidant, 3000 mg; cobalt, 80 mg; copper, 2000 mg; iodine, 400; iron, 1200 mg; manganese, 18,000 mg; selenium, 60 mg, and zinc, 14,000 mg.

Chickens performance measurement

Feed intake and body weight were measured on pen basis. Values of feed intake and body weight were used to calculate feed conversion ratio (FCR). Mortality was recorded if occurred on daily basis.

Blood and muscle collection

Blood (2 ml) was collected from two birds per replicate by cervical dislocation at the end of the study (day 42). Serum was separated and stored at −20°C until analysis. Chickens were defeathered and pectoral muscle was separated from the same bird and stored at −20°C until analysis.

Serum biochemistry

Serum biochemistry such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), total cholesterol, triglycerides, total protein and blood glucose were determined through biochemistry analyzer (Convergys 100) using commercially available kits and the manufactured protocol was followed.

Muscle histomorphology

For muscle histomorphometry, 1 cm² muscle pieces from left superficial pectoral muscle were collected and preserved in 10% formalin solution. These tissues were processed through paraffin embedding technique (Abudabos et al. 2018). The microscopic slides were prepared and stained with H & E. The images of crosssectional muscle fibre tissue were taken at 10× through software using Olympus microscope attached with CCD camera. The average diameter of three muscle fibres from each group was determined by using morphometry software ‘Image J Analysis’. Muscle fibre diameter, Muscle fibre cross sectional Area, muscle fascial diameter and muscle fascial cross sectional area were measured as described by Shah et al. (2019).

Data analysis

Different parameters were analysed through ANOVA using the GLM procedure (SPSS Inc., Chicago, IL). Treatment means are ranked by Tukey’s test as a post-hoc test at P ≤ 0.05. Feed intake, body weight gain and FCR variables were measured on the basis of pen as experimental unit. Bird was used as experimental unit to measure muscle histomorphology and serum biochemistry variables.

Results

The effect of feed additives on feed intake of birds is given in Table 2. The results showed that feed intake was significantly (P < 0.05) higher in coriander (944 g) and protease (944 g) treated birds at the end of the starter phase. Similarly, on overall basis compared to the control, black Cumin (3514 g) and protease (3522 g). No significant difference was found on weekly basis.

Table 2. Impact of addition of phytogenic feed additives and protease in feed on Feed Intake (g) in broilers.

Weeks	Control	Ajwan	Coriander	Black cumin	Protease enzyme	Pooled SEM	P-value
1	119	129	132	127	140	2.39	0.21
2	282	285	286	287	290	2.66	0.06
3	503	505	527	495	515	4.91	0.32
Total starter phase	903^b	919^ab	944^a	908^b	944^a	7.48	0.04
4	770	790	792	743	780	6.24	0.31
5	831	838	849	845	856	6.83	0.11
6	907	925	929	916	941	5.91	0.76
Total finisher phase	2508	2553	2570	2504	2577	14.5	0.11
Overall mean	3410^b	3471^ab	3514^a	3413^b	3522^a	19.6	0.04

Note: Means having different superscripts in the same row show significant difference (P < 0.05).

The effect of different feed additives on body weight is given in Table 3. The results showed that there was no significant (P < 0.05) difference on weekly as well as cumulative starter and finisher phase basis. On overall basis, body weight was significantly (P < 0.05) higher in protease (1853 g), black Cumin (1792 g) and coriander (1767 g) treated birds compared to the control.

Table 3. Impact of addition of phytogenic feed additives and protease on weight gain (g) in broilers.

Weeks	Control	Ajwan	Coriander	Black cumin	Protease enzyme	Pooled SEM	P-value
1	103	112	115	114	124	2.35	0.92
2	233	236	246	244	251	2.91	0.23
3	312	326	321	335	343	4.19	0.06
Total starter phase	648	674	682	694	718	8.25	0.54
4	374	388	385	396	406	4.06	0.65
5	321	333	342	345	359	5.19	0.92
6	343	354	359	358	371	3.20	0.12
Total finisher phase	1038	1075	1085	1098	1135	11.3	0.11
Overall mean	1686^b	1749^b	1767^ab	1792^a	1853^a	19.0	0.03

Note: Means having different superscripts in the same row show significant difference (P < 0.05).

The effect of different feed additives on FCR is given in Table 4. The results revealed that FCR was significantly (P < 0.05) improved in black Cumin (1.32) and protease enzyme (1.32) treated birds compared to the control (1.39) at the end of the starter. Similar trend was observed at the end of the study. No significant difference was found in serum biochemistry between the control and treatment groups (Table 5).

Table 4. Impact of addition of phytogenic feed additives and protease enzyme in feed on feed conversion ratioin broilers.

Weeks	Control	Ajwan	Coriander	Black cumin	Enzyme	Pooled SEM	P-value
1	1.15	1.15	1.15	1.12	1.13	0.01	0.32
2	1.21	1.21	1.16	1.18	1.15	0.01	0.11
3	1.61	1.55	1.64	1.49	1.51	0.02	0.43
Total starter phase	1.39^a	1.3^a	1.39^a	1.32^b	1.32^b	0.01	0.03
4	2.06	2.04	2.06	1.88	1.93	0.03	0.16
5	2.60	2.52	2.49	2.47	2.39	0.04	0.65
6	2.64	2.61	2.59	2.56	2.54	0.02	0.07
Total finisher phase	2.42	2.38	2.37	2.29	2.27	0.02	0.08
Overall mean	2.02^a	1.99^a	1.99^a	1.91^b	1.90^b	0.02	0.02

Note: Means having different superscripts in the same row show significant difference (P < 0.05).

Table 5. Impact of addition of phytogenic feed additives and protease enzyme in feed on serum biochemistry at day 42 in broilers.

Variables	Control	Ajwan	Coriander	Black cumin	Enzyme	Pooled SEM	P-value
Triglyceride (mg/dl)	41.1	40.5	41.4	39.2	39.7	0.44	0.32
Total Cholesterol (mg/dl)	105	107	107	104	107	0.83	0.12
LDL (mg/dl)	37.3	38.5	38.8	38.4	39.8	0.81	0.44
HDL (mg/dl)	56.8	54.6	54.4	61.0	56.6	0.93	0.91
Glucose (mg/dl)	110.7	111.7	112.8	110.7	109.8	0.14	0.11
Total Protein (g/dl)	4.28	4.44	3.95	4.26	4.15	0.13	0.45

Note: Means having different superscripts in the same row show significant difference (P < 0.05).

The effect of different feed additives on serum biochemistry is given in Table 5. No significant difference was found in blood metabolites between the control and treatment groups.

The effect of different feed additives on muscle histomorphology is given in Table 6. The results indicated that protease muscle fibre diameter, muscle fibre cross sectional area, muscle fascial diameter and muscle fascial cross sectional area were significantly (P < 0.05) higher in protease and black Cumin supplemented birds compared to the control.

Table 6. Impact of addition of phytogenic feed additives and protease enzyme in feed on muscle histomorphology at day 42 in broilers.

Variables	Control	Ajwan	Coriander	Black cumin	Protease enzyme	Pooled SEM	P-value
Muscle fibre diameter (mm)	0.058^b	0.060^b	0.062^ab	0.070^a	0.070^a	0.002	0.02
Muscle fibre cross sectional area (mm^2)	0.003^b	0.003^b	0.003^ab	0.004^a	0.004^a	0.000	0.01
Muscle fasical diameter (mm)	1.988^b	2.185^ab	1.950^b	2.353^a	2.337^a	0.077	0.04
Muscle fasical cross sectional area (mm^2)	3.173^b	3.786^ab	3.065^b	4.483^a	4.383^a	0.267	0.03

Note: Means having different superscripts in the same row show significant difference (P < 0.05).

Discussion

To the best of our knowledge, this is the first study which has compared the effect of different herbal plants and protease enzyme on the performance, muscle histology and blood biochemistry in broilers. The results of the present study revealed that feed intake was higher in birds receiving Coriander in comparison with black Cumin in diet at starter and overall period. The use of protease in broiler diet resulted in higher feed intake at starter, finisher and overall production period. The results of the present study are in line with the findings of Khaligh et al. (2011) who reported significantly reduced feed intake in birds receiving feed supplemented with Ajwan at 10 g/kg as compared to the control group. Shroha et al. (2019) also reported similar results where Ajwan supplementation in feed at 2, 4 and 6 g/kg reduced feed intake in broilers. Difference of opinions exists among the authors in relation to the supplementation of different plant origin feed additives on the feed intake in broiler. The conflicting results could be attributed to many factors such as dose and duration of supplementation, anti-nutritional factors, broiler strain, feed composition and environmental factors (Khan et al. 2012d). In the current study, an increase in feed intake was observed in birds fed with protease supplementation. Similar results were also reported in other reports previously (Gracia et al. 2003; Onderci et al. 2006). Exogenous enzymes such as protease are more effective in nutrient utilization and growth through improved digestion (Dosković et al. 2013). Protease neutralizes the anti-nutritional factors and improves the hydrolysis of protein (Ghazi et al. 2002). In addition, it increases the availability and utilization of different amino acids.

Interestingly, the data obtained from present trial showed that inclusion of black Cumin, Coriander and protease enzyme in broiler feed resulted in higher weight gain for the overall production period. The literature shows various studies in agreement with the results of the present study. For instance, Khan et al. (2012c) reported that the inclusion of black Cumin in broiler feed with a level of 2.5% and 5% has increased weight gain in broilers. Similarly, Freitas et al. (2011) reported that the inclusion of protease at 100, 200, 400, 800 and 1600 ppm in broiler feed improved weight gain in broilers. In agreement with our findings, Jang et al. (2007) recorded significantly lower weight gain in coriander supplemented groups in comparison with control. There could be several reasons for increased weight gain in broilers. For instance, in coriander and protease treatments, birds also consumed more feed and therefore gained more weight. Contrary to the expectations, black Cumin increased weight gain despite the fact that they consumed less feed. The findings are important for poultry producers who are interested in using phytogenics. Multiple reasons have been cited for increased weight gain in broiler such as secretion of enzymes, and enhanced absorption of nutrients through intestines (Abd El-Hack et al. 2018). Since FCR of broiler depends upon the corresponding values of feed intake and body weight. Therefore, improved FCR was found in protease and black cumin treated birds. Improved feed efficiency in response to black cumin and protease enzyme has been reported previously in broilers (Guler and Ertas 2006; Freitas et al. 2011).

The meat quality is influenced by the muscle fibre characteristics. Our results indicated that muscle fibre characteristics were improved in black cumin and protease enzyme supplemented birds. In the published literature, we did not find studies for comparison where the phytogenics and protease enzyme have been studied on muscle histology in broiler. Improved meat quality has been reported in broiler fed with black cumin and protease enzyme (Kumar et al. 2017; Disetlhe et al. 2018).

Conclusion

From the results of the present study it was concluded that the performance and muscle histology were significantly improved in protease, black cumin, coriander and ajwain treated broiler.

Acknowledgement

The current study was sponsored by Higher Education Commission of Pakistan (HEC) under Startup Research Grant Programme (SRGP) through project No. 21-1561/SRGP/R&D/HEC/2017.

Disclosure statement

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