Selected herbal plants showing enhanced growth performance, ileal digestibility, bone strength and blood metabolites in broilers

ABSTRACT

The effect of Ajwain, Fenugreek and black Cumin (AFC) in different concentrations on the performance, ileal digestibility, blood metabolites and bone strength of broiler was studied. A total of 360 birds were divided randomly into control (Group A), basal diet + 1.5 g/kg AFC (Group B), basal diet + 3.0 g/kg AFC (Group C), basal diet + 4.5 g/kg AFC (Group D). Feed intake was significantly lower (P < 0.05) in birds fed with AFC at the rate of 4.5 g/kg at starter and finisher phases, however, body weight and feed conversion ratio (FCR) were significantly (P < 0.05) improved in birds fed with AFC at the rate of 3.0 g/kg. Crude protein, crude fat and nitrogen-free extract were significantly (P < 0.05) higher in group fed with 3.0 g/kg AFC. Total cholesterol, triglyceride and low-density lipoprotein (LDL) were significantly (P < 0.05) lower in birds fed with 3.0 g/kg AFC while high-density lipoprotein (HDL) was significantly (P < 0.05) higher in the same group. No significant changes were found in the bone quality parameters between the control and treatment groups. From the results of the present study, we suggested that an herbal mixture of AFC at the rate of 3.0 g/kg improved the body weight, FCR, digestibility and blood profile of broiler.

KEYWORDS:

• Broiler
• performance
• digestibility
• bone strength
• blood profile

Introduction

Different plant species and their extracts can support both health and performance of broiler either individually or as a combined mixture (Khan et al. 2012a; Alzawqari et al. 2016; Stoev et al. 2019; Khoobani et al. 2020). Phytogenics may also possibly replace antibiotics as feed additives (Raza et al. 2016; Ahmad et al. 2020). Herbs may be utilized in broiler feed to ameliorate performance by improving feed properties and remodelling feed quality (Shahid et al. 2015; Safiullah et al. 2019). In addition, different researchers concluded that herbal products contain certain components which quicken growth rate, ameliorate feed consumption and carcass output in broiler (Abudabos et al. 2016; Ali et al. 2019). In addition, other compounds such as essential oils in herbal plants enhance production of digestive enzymes, balance the microbial ecosystem of gut and improve the performance of birds (Alhidary et al. 2017; Zia ur Rahman et al. 2017; Abudabos et al. 2018; Chand et al. 2018).

Ajwain (Carum copticum) has many therapeutic properties and has been used as digestive stimulant. In addition, it has also antibacterial, antifungal, antioxidant and anti-spasmodic properties (Zarshenas et al. 2013). Research studies have documented that Ajwain improved body weight in broiler (Falaki et al. 2016). Khaligh et al. (2011) and Shroha et al. (2019) reported reduced feed intake in response to Ajwain in broiler. Fenugreek (Trigonella foenum-graecum) is considered as a rich energy ingredient and crude protein. Fenugreek promotes animal health, accelerates growth process, boosts up feed conversion ratio (FCR) and increases hunger (Wahab et al. 2019). Fenugreek seeds have curative effects such as anti-helminthic, antibacterial, hypoglycaemic, inflammatory activities and have beneficial effects on digestive system (Dixit et al. 2005). Fenugreek seeds are used as a growth promoter due to the presence of biotin, trimethylamine and neurin, which influence the nervous system and increase hunger (Michael and Kumawat 2003). Fenugreek contains choline and lecithin which are helpful in dissolving fatty matter and cholesterol (Michael and Kumawat 2003). Weerasingha and Atapattu (2013) reported that 4% and 5% Fenugreek reduced the body weight in broiler. El-Mallah et al. (2005) reported that the supplementation of 2% Fenugreek seed powder in turkey feed improved the nitrogen-free extract digestibility.

Black Cumin (Bunium persicum) is a medicinal plant mainly found in Asia and Europe. Black cumin is used for both culinary and medicinal purposes and can correct urinary and digestive disorders. This plant has antifungal properties (Zarshenas et al. 2013) and its seeds have been proved to have therapeutic effects to deal with diarrhoea and dyspepsia (Hassanzad et al. 2018). Sorwar et al. (2016) reported that black cumin leaf supplementation improved the performance of broiler. Most of the studies have reported individual effects of herbal plants in broiler. No study has reported the combined effect of Ajwain, Fenugreek and black Cumin (AFC) in broilers. The aim of the present study was to find the additive effects of the herbal plants in different concentrations on the performance, digestability of nutrients, bone strength and blood metabolites in broiler.

Materials and methods

This study was approved by the committee on ethics and animal welfare, The University of Agriculture, Peshawar, Pakistan.

Animal housing

A total of 360-day-old broilers were purchased from the local market. These birds were divided into four groups and each group contained six replicates with 15 birds. The experiment was conducted in a completely randomized design in cages in an open-sided house. Saw dust was used as bedding material. Initial temperature of the house was fixed at 35°C during the first week and then gradually brought to 25°C during the following week. Birds were fed a starter and finisher ration prepared according to NRC (1994) recommendations (Table 1). Water was available ad libitum and light was maintained for 18 h throughout the experiment.

Table 1. Composition of experimental diet.

Ingredients (%)	Starter diet	Finisher diet
Maize	66.5	67.0
Cotton seed cake	5.00	7.00
Fish meal	9.00	6.50
Wheat bran	15.8	15.0
Methionine	0.10	0.02
Lysine	0.60	0.05
Soya bean oil	2.20	3.13
Salt	0.40	0.40
Lime stone	0.40	0.90
Calculated %
Dry matter	92.2	93.0
Ash	8.40	8.44
Crude fibre	14.3	14.3
Crude protein	21.2	18.2
Ether extract	4.72	4.74

Preparation of Polyherbal preparation

Seeds of Ajwain (Carum copticum), Fenugreek (Trigonella foenum-graecum), black cumin (Bunium persicum) were used after verifying their taxonomic features. Seeds of each plant were dried and then milled separately and mixed in equal ratio to form 1.5, 3 and 4.5 g/kg of feed.

Diets and groups

The diet groups were control (CONT), basal diet + 1.5 g/kg PFA, basal diet + 3.0 g/kg PFA, basal diet + 4.5 g/kg PFA. For determination of apparent ileal digestibility (AID), grower diet was mixed with titanium dioxide (Sigma Aldrich) at the rate of 5 g/kg as indigestible marker.

Measurement of production traits

Feed intake, body weight and FCR of birds were measured per pen basis at weekly interval.

Determination of apparent ileal digestibility

At the end of the experiment, two birds per pen were randomly selected and slaughtered. Carcass was dissected and ileal digesta was collected and stored at −20°C for chemical analysis. After freeze-drying, all the feed and ileal digesta samples were analysed for dry matter, crude protein, ash, crude fat, nitrogen-free extract by the method described by Hafeez et al. (2016) using the formula.$\mathrm{Apparent}\mathrm{ileal}\mathrm{Digestibility}(\text{\%})=100-{\displaystyle \frac{\mathrm{conc}.\mathrm{of}\mathrm{marker}\mathrm{in}\mathrm{feed}}{\mathrm{conc}.\mathrm{of}\mathrm{marker}\mathrm{in}\mathrm{digesta}}\times {\displaystyle \frac{\mathrm{conc}.\mathrm{of}\mathrm{nutrient}\mathrm{in}\mathrm{digesta}}{\mathrm{conc}.\mathrm{of}\mathrm{nutrient}\mathrm{in}\mathrm{feed}}\times 100}}$Chromic oxide was used as an inert marker, which was mixed in the feed three days before the end of the study. Calcium and phosphorus were determined with the help of atomic absorption (PRKIN-ELMER, Connecticut, USA) and spectrophotometer (IRMECO Model U2020 QmbH, Geesthacht, Germany) respectively.

Blood collection and determination of metabolites

The blood samples were collected from 2 birds at day 42 for measuring total cholesterol level, high-density lipoprotein (HDL) cholesterol, triglycerides and low-density lipoprotein (LDL) cholesterol by chemistry analyzer (NSA-300; Neusoft Medical Systems Co., Ltd., Shenyang, China) using commercial kits (Zhongsheng Biochemical Company Beijing, China). Moreover, total blood protein and glucose were also measured with the help of commercial kits (Singapore Bioscience PTE limited).

Bone quality

Two birds from each replicate were slaughtered at day 42. The lift tibia was removed, from which the flash and patella were separated. The bone was cleaned, weighed and frozen for −20°C for further analysis. Tibia quality parameters include weight, length, dry matter and ash was determined. Length was measured by calliper and dry matter was determined through oven at 100°C for 24 h. Ash was calculated by Muffle Furnace at 550°C for overnight.

The Robusticity index and Tibiotarsal index of tibia bone were calculated by the following formula.$\mathrm{Robusticity}\mathrm{index}=\mathrm{bone}\mathrm{length}{/}^3\surd \mathrm{of}\mathrm{bone}\mathrm{weight}$

(Riesenfeld 1972)$\mathrm{Tibiotarsal}\mathrm{index}={\displaystyle \frac{\mathrm{diaphysis}\mathrm{diameter}\text{-}\mathrm{medullary}\mathrm{canal}\mathrm{diameter}}{\mathrm{diaphysis}\mathrm{diameter}}\times 100}$(Barnet and Nordin 1960)

Statistical analysis

Data were analysed through analysis of variance (ANOVA) using statistical software SPSS 21.0 (SPSS Inc., Chicago, IL). Means were separated for the significant difference using the Tukey test. P-value less than 0.05 was considered statistically significant unless otherwise mentioned.

Results

The week wise feed intake was calculated per bird for six weeks in all groups and is demonstrated in Table 2. At the end of starter phase, significantly lower (P < 0.05) feed intake was recorded in group B (897 ± 14.2 g) and D (893 ± 38.8 g) in comparison with Group-C (933 ± 11.2 g). During finisher phase, significantly higher (P < 0.05) feed intake was noted for Group-B (2615 ± 12.5 g) and Group-A (2579 ± 23.2 g) followed by Group-C (2506^b±47.5 g) and Group-D (2442 ± 12.8 g). Overall feed intake was significantly higher (P < 0.05) for Group-A (3596 ± 35.3 g) and Group-B (3513 ± 26.7 g) in comparison with Group-C (3439 ± 58.8 g) and Group-D (3336 ± 52.2 g).

Table 2. Effect of dietary mixture of Ajwain, Fenugreek and black Cumin on feed intake of broilers.

Weeks	A (0 g/kg)	B (1.5 g/kg )	C (3.0 g/kg)	D (4.50 g/kg)	P-value
Starter phase	916^b ± 12.4	897^c ± 14.2	933^a ± 11.2	893^c ± 38.8	0.004
Finisher phase	2579^a ± 23.2	2615^a ± 12.5	2506^b ± 47.5	2442^c ± 12.8	0.005
Overall (day 1–42)	3596^a ± 35.3	3513^a ± 26.7	3439^b ± 58.8	3336^c ± 52.2	0.002

Note: Mean values bearing different superscripts differ significantly (P < 0.05).

The week wise body weight was calculated for six weeks in all groups and is demonstrated in Table 3. During starter phase, weight gain was significantly lower (P < 0.05) in Group-D (601 ± 7.15 g) in comparison with Group-B (735 ± 9.67 g) and C (723 ± 10.9 g). During finisher phase from day 22 today 42, weight gain was significantly higher (P < 0.05) in Group-A (1106 ± 18.9 g) and Group-C (1069 ± 13.5 g) followed by Group-B (1027 ± 17.1 g) in comparison with Group-D (925 ± 10.3 g). For overall period from day 1 to day 42, weight gain was found significantly lower (P < 0.05) for Group-D (1562 ± 17.4 g) in comparison with Group-A (1801 ± 37.3 g), Group-C (1792 ± 24.4 g) and Group-B (1762 ± 26.7 g).

Table 3. Effect of dietary mixture of Ajwain, Fenugreek and Black cumin on body weight of broilers.

Weeks	A (0 g/kg)	B (1.5 g/kg)	C (3.0 g/kg)	D (4.5 g/kg)	P-value
Starter phase	695^b ± 18.36	735^a ± 9.67	723^a ± 10.9	601^c ± 7.15	0.005
Finisher phase	1106^a ± 18.9	1027^b ± 17.1	1069^a ± 13.5	925^c ± 10.3	0.005
Overall (day 1–42)	1801^a ± 37.3	1762^b ± 26.7	1792^a ± 24.4	1562^c ± 17.4	0.002

Note: Similar superscripts in the same row have significantly similar means (P < 0.05).

The FCR was recorded in all groups on weekly basis and is demonstrated in Table 4. During starter phase, significantly lower (P < 0.05) FCR was in Group-B (1.19 ± 0.01) followed by Group-C (1.25 ± 0.01), Group-A (1.26 ± 0.03) and Group-D (1.40 ± 0.02). During finisher phase, FCR was significantly lower (P < 0.05) in Group-A (2.37 ± 0.02) and Group-C (2.41 ± 0.06) in comparison with Group-B (2.54 ± 0.03) while significantly higher FCR was recorded in Group-D (2.68 ± 0.017). For Overall period, significantly higher (P < 0.05) FCR was recorded in Group-D followed by Group-B, Group-A and Group-C.

Table 4. Effect of dietary mixture of Ajwain, Fenugreek and Black cumin on feed conversion ratio of broilers.

Weeks	A (0 g/kg)	B (1.5 g/kg )	C (3.0 g/kg)	D (4.50 g/kg)	P-value
Starter phase	1.26^b ± 0.03	1.19^c ± 0.01	1.25^b ± 0.01	1.40^a ± 0.02	0.005
Finisher phase	2.37^c ± 0.02	2.54^b ± 0.03	2.41^c ± 0.06	2.68^a ± 0.017	0.005
Overall (day 1–42)	1.95^ab ± 0.03	1.99^b ± 0.09	1.91^c ± 0.02	2.18^a ± 0.05	0.004

Note: Similar superscripts in the same row have significantly similar means (P < 0.05).

Table 5 describes the impact of supplementation of AFC in finisher broiler on AID of nutrients in broiler. The apparent digestibility at finisher phase for dry matter and ash remained similar (P > 0.05) after the addition of AFC in feed at different levels. The results showed that AID of crude protein was significantly higher in Group-C (68.4 ± 1.55%) in comparison with Group-A (63.5 ± 2.14%) and Group-D (62.9 ± 1.89%); Moreover, the apparent digestibility of Group-B (66.4 ± 3.06%) was significantly higher than Group-D (62.9 ± 1.89%). The AID of crude fat was significantly higher in Group-C (75.7 ± 1.87%) in comparison with Group-D (72.1 ± 1.84%) with no differences in Group-A (72.9 ± 2.30%) and Group-B (74.5 ± 2.56%). The apparent digestibility of nitrogen-free extract was significantly higher in Group-C (81.0 ± 3.54%) as compared to Group-A (75.9 ± 2.77%) and Group-D (76.1 ± 2.52%) with no changes in Group-B (78.2 ± 2.42%). The apparent digestibility of calcium and phosphorus at finisher phase remained unchanged (P > 0.05).

Table 5. Effect of dietary mixture of Ajwain, Fenugreek and Black cumin on apparent ileal digestibility (%) of nutrients of broiler at finisher phase.

	A (0 g/kg)	B (1.5 g/kg )	C (3.0 g/kg)	D (4.50 g/kg)	P-value
Dry matter	73.7 ± 1.88	73.7 ± 3.03	72.5 ± 3.15	74.3 ± 2.31	0.567
Ash	46.0 ± 2.17	44.6 ± 2.93	43.4 ± 2.48	43.7 ± 1.87	0.143
Crude protein	63.5^bc ± 2.14	66.4^ab ± 3.06	68.4^a ± 1.55	62.9^c ± 1.89	<0.001
Crude fat	72.9^ab ± 2.30	74.5^ab ± 2.56	75.7^a ± 1.87	72.1^b ± 1.84	0.012
Nitrogen free extract	75.9^b ± 2.77	78.2^ab ± 2.42	81.0^a ± 3.54	76.1^b ± 2.52	0.004
Calcium	24.8 ± 1.88	26.2 ± 2.49	26.3 ± 1.97	25.3 ± 2.13	0.458
Phosphorus	23.4 ± 1.66	25.3 ± 2.12	25.5 ± 2.69	24.8 ± 1.90	0.197

Note: Similar superscripts in the same row have significantly similar means (P < 0.05).

Table 6 shows the effect of the addition of AFC in finisher feed on blood parameters in broilers. Triglyceride level was significantly higher in Group-A (37.2 ± 2.91 mg/dl) in comparison with Group-C (32.7 ± 1.60 mg/dl) without affecting Group-B (34.1 ± 3.15 mg/dl) and Group-D (34.9 ± 1.21 mg/dl). The level for total cholesterol was significantly lower in Group-C (110 ± 2.89 mg/dl) than Group-B (116 ± 1.87 mg/dl) without having any changes in Group-A (111 ± 6.97 mg/dl) and Group-D (113 ± 2.65 mg/dl). The level of LDL was significantly higher in Group-A (35.7 ± 1.82 mg/dl) and Group-B (34.2 ± 1.78 mg/dl) than Group-C (31.3 ± 1.65 mg/dl) whereas Group-D (33.4 ± 2.14 mg/dl) was unchanged. The HDL concentration was significantly higher in Group-C (58.6 ± 3.48 mg/dl) as compared to Group-A (53.8 ± 2.60 mg/dl), Group-B (54.8 ± 2.48) and Group-D (53.0 ± 1.72 mg/dl). Glucose and protein content in blood at finisher phase was unchanged (P > 0.05) by the addition of mixture of AFC at 1.5, 3.0 and 4.5 g/kg in the feed.

Table 6. Effect of dietary mixture of Ajwain, Fenugreek and Black cumin on blood parameters of broiler at finisher phase.

	A (0 g/kg)	B (1.5 g/kg )	C (3.0 g/kg)	D (4.50 g/kg)	P-value
Triglyceride (mg/dl)	37.2^a±2.91	34.1^ab±3.15	32.7^b±1.60	34.9^ab ± 1.21	0.007
Total cholesterol (mg/dl)	111^ab ± 6.97	116^a ± 1.87	110^b ± 2.89	113^ab ± 2.65	0.027
LDL (mg/dl)	35.7^a ± 1.82	34.2^a ± 1.78	31.3^b ± 1.65	33.4^ab ± 2.14	0.001
HDL (mg/dl)	53.8^b ± 2.60	54.8^b ± 2.48	58.6^a ± 3.48	53.0^b ± 1.72	0.001
Glucose (mg/dl)	11.8 ± 0.40	11.6 ± 0.73	11.6 ± 0.96	11.9 ± 0.85	0.762
Total protein (g/dl)	3.55 ± 1.50	3.94 ± 0.48	3.88 ± 0.61	3.85 ± 0.37	0.810

Note: Similar superscripts in the same row have significantly similar means (P < 0.05).

Table 7 indicates the effect of supplementation of AFC in broiler finisher feed on various blood parameters. All of the blood parameters under consideration including bone weight, length, dry matter (%), ash (%), robusticity index and tibiotarsal index were not affected (P > 0.05) due to the inclusion of AFC at different levels in broiler finisher ration.

Table 7. Effect of dietary mixture of Ajwain, Fenugreek and Black cumin on bone quality of broile finisher phase.

	A (0 g/kg)	B (1.5 g/kg )	C (3.0 g/kg)	D (4.5 g/kg)	P-value
Weight (g)	13.4 ± 0.96	13.0 ± 1.23	13.6 ± 0.59	13.9 ± 0.88	0.302
Length (cm)	9.59 ± 0.42	9.73 ± 0.49	9.73 ± 0.41	9.71 ± 0.44	0.907
DM (%)	53.6 ± 1.81	54.0 ± 1.59	53.9 ± 2.11	53.0 ± 1.86	0.699
Ash (%)	52.7 ± 1.75	51.5 ± 1.50	51.5 ± 2.29	53.0 ± 1.47	0.203
Robusticity index	4.15 ± 0.07	4.13 ± 0.04	4.15 ± 0.04	4.15 ± 0.04	0.710
Tibiotarsal index	42.0 ± 1.15	41.4 ± 1.71	42.5 ± 1.05	42.0 ± 1.23	0.364

Note: Similar superscripts in the same row have significantly similar means (P < 0.05).

Discussion

In the current experiment, overall feed intake was reduced in treated groups in comparison with control group. However, weight gain was higher in the treatment groups. Similarly, FCR was also improved in the treatment groups compared to the control. Reduced feed intake has been reported previously in experiments conducted on broiler fed with these plants (Duru et al. 2013) or combination of some other herbal products (Ahmadian et al. 2020; Galli et al. 2020). The lower feed intake may be due to the unpleasant smell, taste and anti-proteolytic activities. In the current study, Overall weight gain was recorded significantly (P < 0.05) higher in Group-C and Group-B in comparison with Group-D. Similar results were reported in broiler fed with current regimen of medicinal plants (Mamoun et al. 2014; Valiollahi et al. 2014; Falaki et al. 2016). Diet supplemented with AFC was due to the speedy secretion of amylase, lipase and protease enzymes. In addition, in the presence of the medicinal plants the diet is efficiently utilized resulting in improved feed to gain ratio. In addition, the feed intake was higher in birds fed with 4.5 g/kg AFC, however, corresponding weight gain was reduced. The results indicated that higher dose (4.5 g/kg) did not produce linear results, probably the higher dose was not appropriate for enhanced performance in broiler.

In the current study, the combination of plants at the rate of 3 g/kg increased the crude protein, crude fat and nitrogen free extract in broiler at the end of the experiment. Apparently, the improved profile of these contents is the major reason for higher weight gain in the respective group. Fenugreek contains minerals, B-complex vitamins, iron, para amino benzoic acid, vitamin A and D, phosphates, lecithin and choline which have the ability to dissolve the fatty acids and cholesterol (Dixit et al. 2005). Moreover, the Fenugreek has the ability to increase the appetite by exerting pressure on the nervous system due to the presence of neurine, biotine and trimethylamine (Michael and Kumawat 2003). In addition to Fenugreek, Carum copticum is another plant used in the present study which is composed of 6 major chemical compounds which include 47% thymol, 32.8% γ-terpinene, 15.2% p-cymene, 2.0% b-pinene, 0.6% myrcene and 0.7% limonene (Hassanzad et al. 2018). The seeds of Carum copticum were frequently used for medicinal purposes to potentiate the digestive processes and for the treatment of some liver problems (Ishikawa et al. 2001). The medicinal properties of Carum copticum include antifungal, antispasmodic, antioxidants and antimicrobial (Zarshenas et al. 2013), which could have contributed to better digestibility and blood profile at the starter phase in the current study. The black cumin seeds have consisted of p-Mentha-1, 4-dien 7-al gamma-terpinene, beta pinene and cuminaldehyde. These plants have much different beneficial effects including digestive, urinary, gynecological problems, diuretics, anti tremor, anti-helminthics, anti-asthma and dyspnoea, which could have exerted beneficial effects in the present study on improved AID in broilers. The exact mechanism of AFC is still not known, however, it is speculated that the higher digestibility of nutrients may be due to the effect that herbal plants have enhanced effects on nutrient digestibility, antimicrobial action, intestinal mucus secretion, release of digestive enzymes and gut morphology.

In the current study, triglyceride, total cholesterol and LDL were significantly decreased while HDL was significantly increased in birds fed with plants at the rate of 3.0 g/kg. The improved blood profile may be due to the presence of vitamins and minerals phosphates, lecithin and choline leading to the lower level of cholesterol profile. Similarly, Carum copticum can lower the triglycerides and total cholesterol level and improve HDL (Samani and Farrokhi 2014). The exact mechanism of the action of the medicinal plants in lowering the lipid profile is not fully understood. It is usually thought that these herbal plants interfere with cholesterol biosynthesis in liver and the conversion of cholesterol into bile acid (Khan et al. 2012a). In addition, the herbal plants also inhibit HMG-CoA reductase and fatty acid synthase which are rate-limiting enzymes of liver (Khan et al. 2012b).

Conclusion

The results showed that feed intake was significantly lower in group of birds fed with AFC at the rate of 4.5 g/kg, while body weight and FCR were significantly higher in birds fed with AFC at the rate of 3.0 g/kg. Similarly, digestibility of crude protein, fat and nitrogen-free extract was significantly higher in AFC at the rate of 3.0 g/kg. In addition, blood metabolites such as triglyceride and total cholesterol were significantly lower and HDL concentration was significantly higher in birds fed with AFC at the rate of 3.0 g/kg.

Acknowledgements

We are thankful to Prof. Dr. Sarzamin Khan, Dr. Naila Chand and Mr. Muhammad Hanif Khan for helping in conducting this expeirment.

Disclosure statement

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