Effects of graded dose dietary supplementation of Piper betle leaf meal and Persicaria odorata leaf meal on growth performance, apparent ileal digestibility, and gut morphology in broilers

Abstract

Due to antimicrobial resistance and the public health hazard of antibiotic growth promoters, there is a grave need to find potential alternatives for sustainable poultry production. Piper betle (PB) and Persicaria odorata (PO) are herbs, which have been reported for antimicrobial, antioxidant, and anti-inflammatory properties. The present study aimed to estimate the influence of different dose supplementation of Piper betle leaf meal (PBLM) and Persicaria odorata leaf meal (POLM) on growth performance, ileal digestibility and gut morphology of broilers chickens. A total of 210 one day-old broiler chicks were randomly grouped into 7 treatments, and each treatment group has 3 replicates (n = 10) with a total number of 30 chicks. The treatments included T₁ control (basal diet (BD) with no supplementation), T₂ (BD + 2 g/kg PBLM); T₃ (BD + 4 g/kg PBLM), T₄ (BD + 8 g/kg PBLM), T₅ (BD + 2 g/kg POLM), T₆ (BD + 4 g/kg POLM), T₇ (BD + 8 g/kg POLM). Growth performance, gut morphology and ileal digestibility were measured. Except for T₄ (8 g/kg PBLM), graded dose inclusion of PBLM and POLM increased (P < 0.05) the body weight gain (BWG), positively modulated the gut architecture and enhanced nutrient digestibility in both stater and finisher growth phases of broiler chickens. Birds fed on PBLM 4 g/kg (T₃), and POLM 8 g/kg (T₇) had significantly higher (P < 0.05) BWG with superior (P < 0.05) feed efficiency in the overall growth period. Chickens fed on diets T₃ and T₇ had longer (P < 0.05) villi for duodenum as well as for jejunum. Furthermore, the birds fed on supplementations T₃ and T₇ showed improved (P < 0.05) digestibility of ether extract (EE), and dry matter (DM) compared to the control group. However, least (P < 0.05) crude protein (CP) digestibility was recorded for T_4. In conclusion, dietary supplementations of PBLM 4 g/kg and POLM 8 g/kg were positively modulated the intestinal microarchitecture with enhanced nutrient digestibility, resulted in maximum body weight gain, thus improved the growth performance of broiler chickens.

1. Introduction

For decades, the sub-therapeutic inclusion of antimicrobial growth promoters (AGPs) has been successfully and routinely practised in broiler chicken’s production. The AGPs increase the growth performance of birds by improving their intestinal health, enhancing nutrient absorption and utilisation. The inclusion of AGPs as a feed additive is the most common and economical way to improve poultry performance (Debnath et al., 2014). However, the use of AGPs has resulted in antimicrobial resistance and accumulation of residues in edible tissues, that may increase risks to human health (Cosby et al., 2015). This danger of antibiotic resistance has led to the ban against sub-therapeutic use of AGPs in food-producing animals (Toghyani et al., 2011). The threat of antibiotic-resistant pathogens leads to a growing dynamic in research and development of future alternative growth promoters for animal production (Al-Abd et al., 2015). Many alternatives to AGPs are introduced, while recently phytogenic feed additives (PFAs) gained consideration as a potential alternative in poultry production (Murugesan et al., 2015).

Natural substances like plants, herbs, plant extracts, essential oils, and spices, are referred to as PFAs or phytobiotics. Recently the phytobiotics got attention as potential feed additives in poultry production (Abudabos et al., 2016). The PFAs have numerous positive effects on poultry nutrition, which includes better feed intake, higher digestibility, and increased plasma total antioxidant capacity. They are potent antimicrobial, coccidiostats, anthelmintic, and immune-stimulating substances (Manafi, 2015). Supplementation of PFAs increases the apparent total tract nutrient digestibility (Murugesan et al., 2015) as well as improvement in the gut morphology of broilers (Ahsan et al., 2018), with a potential to favour the over-all health of broiler chickens (Paraskeuas et al., 2017). Various studies showed that PFAs might increase feed efficacy in broilers, as PFAs or phytobiotics, thinning the mucosal surface of gut resulted in increased diffusion of nutrients from the apical surface of epithelial cells, similar to AGPs (Yang et al., 2015). Many researchers are investigating phytogenic plants, including natural herbs, in terms of their nutraceutical, antimicrobial, and nutritive potential. Among such herbs of importance are Piper betle and Persicaria odorata.

The Piper betle belongs to the family Piperaceae, an essential herbal plant having significant medicinal value and variety of pharmacological activities. It is distributed throughout East Africa and the tropical regions of Asia and commonly known as “daun sirih”. The Piper betle leaves possess many valuable bioactivities and have been used in traditional medicinal systems (Patra et al., 2016). The Piper betle has been reported as potent antimicrobial (Syahidah et al., 2017), antioxidant (Jaiswal et al., 2014) and anthelmintic (Shah et al., 2016) herb. Previous phytochemical investigations reported the presence of high phenolic compounds in Piper betle; however, the reported significant bioactive compounds were hydroxychavicol and eugenol, and these bioactive compounds have been studied and reported as potent antimicrobial agents (Atiya et al., 2018, Syahidah et al., 2017).

The Persicaria odorata belongs to the family Polygonaceae, and it is also called as Polygonum minus Huds. The Persicaria odorata routinely used in Southeast Asian cuisine as a natural and traditional herb, and it is commonly named as “daun kesum” or “daun laksa” (Vikram et al., 2014). Previous studies shown that Persicaria odorata is a potent antioxidant (Cristapher et al., 2016), contains flavonoids and a high concentration of essential oils (Narasimhulu et al., 2014). Antiviral, antifungal, and antimicrobial activities of Persicaria odorata has also been reported (Vikram et al., 2014, Christapher et al., 2015). The said major bioactive compounds of Persicaria odorata include quercetin and myricetin. In poultry, quercetin has shown antiviral and antioxidant effects, additionally have a potential role in counteracting heavy metal toxicity (Saeed et al., 2017).

Very few studies are reported previously on the dietary inclusion of herbs in broiler chickens which contains tannin, as earlier it was assumed that tannin has anti-nutritional effects in monogastric animals. However, from the last few years, various studies revealed that the dietary supplementation of several tannin sources in low concentrations resulted in improved health status, thus enhanced the growth performance by positively modulating the gut and its eco-system in mono-gastric animals (Brus et al., 2013, Starčević et al., 2015). The Piper betle and Persicaria odorata have not been investigated in broiler chickens (to the best of our knowledge) for their effects on growth performance measures, gut health, and nutrient digestibility. Taking into account the medicinal potential of Piper betle and Persicaria odorata, this study has been anticipated to evaluate the influence of gradual level inclusion of PBLM and POLM on growth performance, gut morphology, and apparent ileal digestibility in broiler chickens.

2. Materials and methods

2.1. Ethical approval

Institutional Animal Care and Use Committee, Universiti Putra Malaysia approved the animal handling procedures for this experiment (UPM/IACUC/AUP-R033/2018).

2.2. Source and preparation method for diet

Fresh samples of Piper betle (PB) and Persicaria odorata (PO) were collected from herbarium garden, Universiti Putra Malaysia. The plant leaves were validated from Unit Biodiversity, Institute of Bio-Sciences, University of Putra Malaysia. Fresh sample (leaves) of both herbs were deposited with voucher no. SK3294/18 and SK3296/18 respectively for future reference. The collected leaves were subjected to dry at 50 °C for 72 h and pulverised to a fine powder. The prepared plant samples (fine powder) stored at 4 °C until further usage.

2.3. Management of birds

Two hundred and ten one day-old broiler chicks (Cobb 500) were procured from a local hatchery and individually wing-banded upon arrival. After being weighed, the chicks were randomly assigned into seven treatment groups; each treatment group consisted of 3 replicates of 10 chicks (n = 30). The chickens were raised into battery cages in an open-sided house, with free access to water and feed throughout the trial period. The cyclic temprature of the house were (maximum 34°C; minimum 24) and humidaty (maximum 91%; minimum 61%). Chicks were vaccinated with the following schedule:

• i)ND and IB (Newcastle disease and Infectious bronchitis) at the age of day 4 and day 21, administered via intra-ocular route.
• ii)IBD (Infectious bursal disease) at the age of day 7, administered via intra-ocular route.

2.4. Experimental design and supplemented treatments

The birds were raised on the starter diet during 0 to 21 days and finisher diet during 22–42 days. Treatments were: T₁ (basal diet (BD) with no supplementation) control, T₂ (BD + 2 g/kg PBLM); T₃ (BD + 4 g/kg PBLM), T₄ (BD + 8 g/kg PBLM), T₅ (BD + 2 g/kg POLM), T₆ (BD + 4 g/kg POLM), T₇(BD + 8 g/kg POLM). The supplemented experimental diets meet or exceed the NRC (1994) recommendations, presented in (Table 1). Moreover, the basal diet was without any premixing of anticoccidial, antimicrobial, and antioxidant drugs or feed enzymes.

Table 1.

Ingredient (%age as feed) and nutrient composition of the basal diet.

Ingredients (%)	Starter	Finisher
Corn	54.40	58.95
Soybean meal (SBM) (44%)	33.90	28.00
Fish Meal	5.33	5.74
Palm oil	2.64	4.14
Limestone	1.06	0.86
Salt	0.38	0.28
Dicalcium Phosphate	1.09	0.84
Mineral Mix*	0.28	0.28
Vitamin Mix**	0.29	0.29
Choline chloride	0.10	0.10
L-Lysine	0.35	0.34
DL-Methionine	0.18	0.18
Calculated analysis
(ME) MJ/kg	13.10	13.40
Crude Fat, %	5.21	7.01
Crude Protein, %	22.00	20.00
Calcium, %	0.99	0.90
Available P, %	0.43	0.35

^*Premix supplied minerals per kg of dietary feed: Cu, 19.99 (mg); Zinc, 100.01 (mg); Mg, 16.0 (mg); iron, 120.0 (mg); I, 0.8 (mg); Co, 0.6 (mg).

^**Premix supplied vitamins per kg of dietary feed: Vitamin E, 0.02 (mg); Vitamin D3; 1200 (IU); Vitamin A, 6500 (IU); VitaminB1, 0.83(mg); riboflavin, 2.0(mg); Vitamin K (menadione) 1.33(mg); Vitamin B12, 0.03(mg); Vitamin B3 24 (mg); Vitamin B6, 1.37 (mg); Biotin, 0.03 (mg); Calcium D-Panthothenate, 3.69 (mg); Folic acid, 0.33 (mg).

2.5. Sample collection

Every week during the whole experiment, the live body weight (BW) and feed intake (FI) were recorded for each bird to measure growth performance. Furthermore, body weight gain (BWG) and feed conversion ratio (FCR) were calculated. On day 21 six chickens (n = 2 per replicate) and day 42 twelve chickens (n = 4 per replicate) were selected randomly, and slaughtered according to the procedure as described in “The Malaysian Standard (MS) 1500: 2009”. The intestinal samples were obtained to determine the gut morphology (villi height and crypt depth), while ileal digesta samples were collected to measure apparent ileal digestibility as described by (Alshelmani et al., 2016).

2.6. Gut morphology

The intestinal morphology was studied in line with the procedure described by (Alshelmani et al., 2016). About 5–6 cm long fragments of ileum, jejunum and duodenum were collected. The ileum segments were harvested (midway between the Meckel’s diverticulum to the anterior part of the ileocaecal junction). While the sections from the jejunum were collected (midway between the distal portion of the duodenal loop and Meckel's diverticulum), and duodenum segments were collected (pylorus to the distal part of the duodenal loop). A physiological solution was used for flushing intestinal fragments, and a 10% buffer formalin solution was used to fix the sample. The intestinal pieces were cut approximately up to 3.5 mm and then subjected to process, embedding with paraffin and stained using “hematoxylin and eosin” staining technique. The twelve villi and crypt unit were used for all observations. Furthermore, the morphometric parameter of each segment of intestine, including villus height (VH) and villus crypt depth (CD) were viewed under a light microscope installed with a digital camera (Leica DFC 295, Wetzlar, Germany). The measurement of villus height was performed from the villus tip to villus crypt junction, while the depth of invagination between two villi consider as crypt depth. For the measurement of villus height and crypt depth, the Image-Pro Plus software was used as described by Touchette, et al. (2002).

2.7. Nutrient digestibility

To determine the apparent ileal digestibility (AID), an indigestible marker “Titanium dioxide (TiO₂)” was added in each treatment group at the dose rate of 3 g / kg of feed. The Marker (TiO₂) was added in diets of broiler chickens on day18 to day 21 during the starter phase and on day 39 to day 42 of age during the finisher phase as described by (Alshelmani et al., 2017). After slaughtering the ileal digesta samples were collected (lower half of ileum) and kept at −80 °C until lab analysis. For digestibility estimation, digesta samples were freeze-dried, then dried in an oven at 80 °C to get constant weight, and finally homogenised. For determination of the dry matter (DM), ether extract (EE), crude protein (CP), and ash contents of digesta and feed; the method “proximate analysis of nutrients” was used as described by (AOAC, 1995). For determination of “TiO₂” concentration of digesta and feed, the homogenised ileal digesta samples were ashed, then digested using sulphuric acid (7.4 M) and subjected to react with “H₂O₂”, and the absorbance was measured at 410 nm using a spectrophotometer (short et al., 1996).

$$\text{AID of nutrient}=100-\left[\left(\%\text{Ti}{\text{O}}_2\text{in feed}/\%\text{Ti}{\text{O}}_2\text{in ileal content}\right)\times \left(\%\text{of nutrient in ileal content}/\%\text{nutrient in feed}\times 100\right)\right]$$

2.8. Statistical analysis

The data were examined for their conformance to the normal distribution prior to analysis using the Kolmogorov-Smirnov test. The data were analysed as a 3 × 2 fractional arrangement with 3 levels of PBLM and POLM. The data were subjected to ANOVA using the general linear model, and the analysis was carried out with SAS (9.4) 2012 (SAS Institute Inc., Cary, NC, USA). The differences between the means of treatments were compared using the LSD, while the overall significance level was set at a value of P < 0.05.

3. Results and discussion

3.1. Growth performance

The influence of graded dose inclusion of PBLM and POLM on the growth performance measures of broiler chickens are presented in (Table 2). The main effects data on day 21 and day 42 showed that BWG, FI and FCR were not influenced with increased concentration of supplements dose. However, a significant interaction was observed for BWG (P < 0.05), indicating higher BWG with increased supplements dose. On day 21 increased (P < 0.05) body weight gain (BWG) was recorded in the chickens fed on supplemented diets T₃ 690.50 g and T₇ 688.10 g compared to the broiler chickens raised on other supplemented diet T₄ and control diet. In the starter phase, dietary supplementation did not influence the FI in all experimental broilers, except those chickens, that fed on supplemented diet T₄, thus resulted in significantly (P < 0.05) low FI. Also, superior feed efficiency was recorded in broilers fed on diets T₃ and T_7, with significantly reduced (P < 0.05) FCR 1.49 and 1.51, respectively, relative to other treatment groups and the control group.

Table 2.

Effects of different dose supplementation of PBLM and POLM on the growth parameter of broilers.

	1–21 days			21–42 days			1–42 days
Treatments	BWG (g)	Feed intake (g)	FCR	BWG (g)	Feed intake (g)	FCR	BWG (g)	Feed intake (g)	FCR
T1	638.39b	1022.59a	1.60a	1639.93b	3002.05a	1.83a	2277.32c	4024.83a	1.76 a
T2	657.82 ab	1033.20 a	1.57 a	1692.46 ab	3019.20 a	1.78b	2350.28b	4052.40 a	1.72b
T3	690.50 a	1030.37 a	1.49b	1741.05 a	3018.20 a	1.73b	2431.55 a	4048.57 a	1.67c
T4	633.04b	981.14b	1.55 a	1655.21b	2965.2b	1.79b	2288.85c	3946.35b	1.72b
T5	659.02 ab	1035.20 a	1.57 a	1690.53 ab	3026.0 a	1.79b	2349.49b	4061.20 a	1.73b
T6	670.70 ab	1031.40 a	1.54 a	1694.33 ab	3023.4 a	1.78b	2365.03b	4054.80 a	1.71b
T7	688.10 a	1038.80a	1.51b	1733.10 a	3013.8 a	1.74b	2421.20 a	4052.60 a	1.67c
SEM	5.9	6.61	0.02	9.34	6.74	0.01	13.62	10.66	0.01
Main effects!
Supplement dose
2 g / kg	658.42	1034.20	1.58	1691.50	3022.60	1.79	2349.91b	4056.80 a	1.73
4 g / kg	680.60	1030.89	1.52	1717.69	3020.80	1.76	2398.29 a	4051.69 a	1.69
8 g / kg	660.57	1009.97	1.53	1694.46	2989.50	1.77	2355.03ab	3999.47b	1.70
Supplements
P. betle	660.45	1014.91	1.54	1696.44	3000.87	1.77	2356.90	4015.77	1.71
P. odorata	672.61	1035.13	1.54	1705.99	3021.07	1.77	2378.59	4056.20	1.71
Source of variation
Supplement dose	0.251	0.335	0.396	0.282	0.107	0.493	0.067	0.048	0.136
Supplements	0.308	0.167	0.955	0.512	0.154	0.862	0.230	0.051	0.965
Supplement × Supplement dose	0.041	0.196	0.592	0.006	0.358	0.117	0.001	0.082	0.037

^abcThe means with different superscripts within the same columns are significantly different (P < 0.05).

T1: (Basal diet (BD); control); T2:(BD + PBLM 2 g/kg); T3:(BD + PBLM 4 g/kg); T4:(BD + PBLM 8 g/kg); T5: (BD + POLM 2 g/kg); T6: (BD + POLM 4 g/kg); T7: (BD + POLM 8 g/kg)

^!Data were analysed as a 3 × 2 factorial arrangement, excluding control group.

The present findings were comparable with the outcomes of Aroche et al. (2018) who identified that feed supplementation of medicinal plants in the form of mix powder including; A. occidentale, P. guajava, and M. citrifolia enhanced the feed efficiency and resulted in superior FCR in starter phase. The reported results might be due to the potential of natural products containing useful bioactive compounds like eugenol and quercetin, which have the potential to improve the biological development of chickens. The secondary metabolites of herbs such as alkaloids, tannins, and flavonoids positively influence the birds' health, as they possess antimicrobial, anti-inflammatory, and antioxidant properties (Perez-Gregorio et al., 2014). Moreover, Aroche et al. (2018) reported that dietary inclusion of P. guajava and M. citrifolia leaf meal resulted in better utilisation of the nutrients with lower feed intake and high growth in the first growth phase of chickens. Further, he emphasised that P. guajava and M. citrifolia were reported antimicrobial and antioxidant (Ali et al., 2016); thus, their inclusion as a dietary supplement helps to improve the growth in chickens. The present study has shown that the BWG of broilers increased with an increasing level of POLM; a similar pattern was observed in PBLM supplemented treatments up to an inclusion level of 4 g/kg of feed. However, the further increase in PBLM (8 g/kg) resulted in significantly decreased BWG. This reduction in BWG at a higher inclusion level of PBLM might be due to high tannin concentrations. Excessive tannins may cause metabolic disturbances like precipitation of protein, that resulted in an anti-nutritional effect (Savón et al., 2006). It is assumed that the higher tannin concentrations resulted in lesser feed intake, and inhibiting the absorption of nutrients, ultimately leads to a decline in growth (Mansoori et al., 2015).

On day 42, the birds fed on diets T₃ and T₇ had maximum (P < 0.05) BWG relative to other supplemented birds of treatment T₄ and the control group. Additionally, there was no influence (P > 0.05) of dietary inclusion of PBLM and POLM on FI; however, FI was recorded least (P < 0.05) for T₄. The FCR was noted maximum (P < 0.05) in T₁ 1.83 in comparison with other dietary treatments, whereas no difference (P > 0.05) was recorded for FCR within supplemented groups. The increased BWG in broilers with PBLM and POLM supplementation might be due to the presence of polyphenols and flavonoids, primarily due to the secondary bioactive compound eugenol and quercetin, which were successfully quantified from PBLM and POLM, respectively (unpublished data). The secondary bioactive compound eugenol has been considered as an appetite stimulant (Kumar et al., 2014), and antioxidant (Kim et al., 2013), it can trigger the function of pancreatic and digestive enzymes (Pradhan et al., 2014). In a study, Paraskeuas et al. (2017) described the potential of eugenol, who reported that the supplementation of phytogenic feed additive including menthol, anethol and eugenol at 100 and 150 mg/kg in broiler chickens enhanced the growth performance, increased digestibility, improved serum total antioxidant capacity and reduced expression of pro-inflammatory cytokine (IL-18). On the other hand, bioactive compound quercetin is a flavone, that can improve the animal growth by up-regulating the growth hormone along with the hepatic growth hormone receptor, this stimulus increases the concentration of insulin-like growth factor-1 (Ouyang et al., 2016). Generally, the dietary supplementation of flavonoids in broiler chickens ration has potential to enhance growth performance of chickens (Starcevic, 2015). In another study, the role of a secondary bioactive compound like quercetin has been revealed by Kim et al. (2015), who narrates that the dietary inclusion of quercetin in broilers has positively improved the growth performance. The quercetin can limit the effects of oxidative stress (Ma et al., 2015), and pro-inflammatory cytokines such as TNF-α, cyclooxygenase-2 interleukin 6 and cyclooxygenase-2 (Zou et al., 2015). The highest BWG was recorded in broilers fed on diet T3 (PBLM 4 g/kg) with superior FCR of 1.73 in the finisher phase. Whereas, further increase in supplementation of PBLM at the dose of 8 mg/kg leads to decline (P < 0.05) in BWG, and least FI 2965.2 g. This decline in BWG and least FI might be due to high tannins concentration. These results endorsed the outcomes of Bee et al. (2017) who revealed that an increased level of chestnut tannins in the feed from 0.71% to 1.5%, resulted in reduced feed intake; thus, declined growth performance in chickens. Similarly, Oso et al. (2019) reported that the supplementation of phytogenic blend containing Piper betle, Cynodon dactylon, and Piper nigrum in broilers, resulted in better FCR and BWG in the treated group fed on 1% phytogenic blend compared to the broilers fed on 2% phytogenic blend. In monogastric animals especially in poultry, the positive influence of tannins depends on the balanced dose or inclusion level. Inclusion of higher tannin concentrations negatively affects the feed palatability. On the other hand, appropriate addition of tannins improves nutrient digestion through protein and enzyme complexation; also, tannins being a potent antimicrobial and antioxidant substances can positively modulate the gut ecosystem, thus enhance the growth performance (Huang et al., 2018). In terms of overall growth performance 1–42 day, the main effects data indicate that, as the dose of the supplement increased, BWG (P < 0.05) and FI (P < 0.05) increased; however, FCR was not affected with increased concentration of supplements. Furthermore, significant interaction was observed for BWG (0.000) and FCR (P < 0.05) highlighted that increased supplements‘ dose resulted in enhanced BWG with reduced FCR, indicating improved feed efficiency. The overall growth performance over 42 days shown that the supplemented diet T₃ achieved the highest BWG 2431.55 g and superior feed efficiency with FCR 1.67 followed by T₇ with BWG 2421.20 g that was maximum (P < 0.05) compared to other supplemented groups and recorded least (P < 0.05) in control diet T₁ with BWG 2277.32 g. In general, phytobiotics treated groups showed significantly higher BWGs compared to the control group. Except for dietary group T_4, supplementation did not influence the FI. The maximum (P < 0.05) FCR was noted in control group T₁ 1.76 relative to supplemented dietary treatments. While significantly least and better (P < 0.05) FCR was recorded for both T₃ and T₇. These results are parallel to Mpofu et al. (2016), who revealed that inclusion of phytobiotics like L. javanica at an inclusion level of 5 g/kg in broilers ration, has a positive impact on overall growth. In another study, Paraskeuas et al. (2017) highlighted similar results, who designated that inclusion of phytogenic feed additive including eugenol, menthol, and anethol at 100 to 150 mg/kg in feed, enhanced nutrient digestibility and thus enhanced growth measures in broiler chickens. Similarly, Salami et al. (2015) identified that the incorporation of medicinal herbs as a feed additive in the broiler’s diets improved the FCR in the last growing phase of birds.

3.2. Gut morphology

For the determination of nutrient response and gut health, the microscopic structure and status of the intestinal tract are considered good indicators (Viveros et al., 2011). The results of intestinal morphometrical parameters on day 21 and day 42 are presented in (Tables 3 and 4). On day 21, the main effects data highlighted that increased in supplements dose significantly (P < 0.05) increased the VH of duodenum and jejunum; additionally, increased the VH: CD of jejunum. However, no significant effect of increased supplements dose was observed for duodenum CD and VH: CD and jejunum CD. Also the VH, CD and VH: CD for ileum, were not influenced by increased supplements dose. Furthermore, Significant interaction was observed for duodenum and jejunum VH (P < 0.0001) and VH: CD (P < 0.05), that shown increased supplement dose increased the VH and VH: CD, in turn, resulted into improved gut health. The inclusion of different levels of PBLM and POLM in feed of birds resulted in an increased VH of all segments of the intestine. On day 21, supplemented diets T₃ and T₇ had increased (P < 0.05) VH in duodenum and jejunum as compared to other supplemented treatments and control group, followed by T₂ and T₆ that have longer (P < 0.05) villi of duodenum and jejunum compared to T₅, T₄, and control T₁. Additionally, no differences (P > 0.05) were recorded in the villus height of ileum among supplemented and the control groups. The dietary inclusion has no influence (P > 0.05) on CD of the duodenum, while the chickens fed supplemented diet T₃ shown least (P < 0.05) CD of jejunum as well as for ileum. The VH: CD of the duodenum as well as for ileum were recorded higher (P < 0.05) in all dietary treatments relative to the control group. While VH: CD of jejunum was observed higher (P < 0.05) in diets T₃, T₇, T₆ and T₂ followed by T₅, T₄, and the control T₁.

Table 3.

Gut morphometric measures of broilers supplemented with different levels of PBLM and POLM on day 21.

	Villus height; (μm)			Crypt depth; (μm)			VH: CD
Treatments	Duodenum	Jejunum	Ileum	Duodenum	Jejunum	Ileum	Duodenum	Jejunum	Ileum
T1	988.34c	686.07c	447.88	136.75	106.57 a	94.34 a	7.25b	6.47b	4.76b
T2	1044.07b	720.61b	459.85	139.06	106.89 a	90.88 a	7.52 a	6.76 a	5.09 a
T3	1097.63 a	759.73 a	464.79	128.25	99.92b	85.29b	8.6 a	7.72 a	5.47 a
T4	1001.91c	680.25c	448.66	136.76	112.36 a	91.18 a	7.34 ab	6.14b	4.95 ab
T5	1008.23c	695.84c	458.66	134.46	110.3 a	89.67 a	7.43 a	6.31b	5.14 a
T6	1018.04 bc	726.99b	454.63	136.14	104.77 a	90.95 a	7.45 a	6.96 a	5.02 a
T7	1084.45 a	756.15 a	464.83	128.18	105.28 a	86.45b	8.36 a	7.25 a	5.41 a
SEM	9.06	4.55	3.36	2.03	1.56	2.06	0.15	0.13	0.16
Main effects!
Supplement concertation
2 g / kg	1026.15b	708.2245b	459.26	137.11	108.55	90.33	7.61	6.66b	5.29
4 g / kg	1057.89 a	743.36 a	459.71	132.24	102.45	88.27	8.20	7.37 a	5.51
8 g / kg	1043.18 ab	718.204b	456.75	133.17	109.32	88.92	8.01	6.69 ab	5.37
Supplements
P. betle	1047.87	720.20	457.76	134.92	106.46	89.28	7.93	6.92	5.36
P. odorata	1036.94	726.33	459.38	133.43	107.08	89.06	7.95	6.90	5.42
Source of variation
Supplement dose	0.015	<0.0001	0.937	0.658	0.211	0.931	0.369	0.070	0.887
Supplements	0.209	0.323	0.824	0.746	0.856	0.960	0.973	0.950	0.865
Supplement × Supplement dose	<0.0001	<0.0001	0.326	0.361	0.325	0.647	0.040	0.019	0.677

^abc The means with different superscripts within the same columns are significantly different (P < 0.05).

T1: (Basal diet (BD); control); T2:(BD + PBLM 2 g/kg); T3:(BD + PBLM 4 g/kg); T4:(BD + PBLM 8 g/kg); T5: (BD + POLM 2 g/kg); T6: (BD + POLM 4 g/kg); T7: (BD + POLM 8 g/kg).

^!Data were analysed as a 3 × 2 factorial arrangement, excluding control group.

Table 4.

Gut morphometric measures of broilers supplemented with different levels of PBLM and POLM on day 42.

	Villus height; (μm)			Crypt depth; (μm)			VH: CD
Treatments	Duodenum	Jejunum	Ileum	Duodenum	Jejunum	Ileum	Duodenum	Jejunum	Ileum
T1	1372.94c	967.31c	645.12	185.11 a	140.78	135.82 a	7.44b	6.88b	4.76b
T2	1440.3b	1035.79 a	665.92	179.72 ab	141.07	130.29 a	8.05b	7.37 ab	5.12b
T3	1483.52 a	1041.68 a	671.49	168.81b	137.05	121.33b	8.81 a	7.61 a	5.54 a
T4	1393.84c	970.04c	649.11	184.43 a	137.9	132.06 a	7.57b	7.04 ab	4.92b
T5	1433.69b	998.03b	666.04	183.72 a	139.87	131.57 a	7.82b	7.15 ab	5.07b
T6	1439.21b	1001.53b	668.55	181.68 ab	138.58	132.53 a	7.94b	7.24 ab	5.06b
T7	1473.11 a	1039.47 a	670.5	168.94b	139.57	122.9b	8.73 a	7.46 ab	5.46 a
SEM	12.29	4.28	2.67	1.74	1.31	1.33	0.1	0.08	0.06
Main effects!
Supplement concertation
2 g / kg	1437b	1017.11 ab	665.96	181.47	140.47	131.33	7.98	7.30	5.11
4 g / kg	1461.37 a	1021.70 a	670.06	175.24	137.46	127.03	8.41	7.48	5.32
8 g / kg	1433.48b	1004.81b	659.81	176.68	138.74	127.08	8.19	7.30	5.24
Supplements
P. betle	1439.22	1015.87	662.16	177.65	138.54	128.06	8.18	7.39	5.21
P. odorata	1448.67	1013.21	668.39	177.95	139.24	128.90	8.21	7.33	5.23
Source of variation
Supplement dose	0.002	0.039	0.309	0.347	0.720	0.277	0.248	0.677	0.443
Supplements	0.157	0.626	0.257	0.936	0.818	0.738	0.862	0.751	0.913
Supplement × Supplement dose	<0.0001	<0.0001	0.150	0.008	0.907	0.006	0.001	0.183	0.012

^abcThe means with different superscripts within the same columns are significantly different (P < 0.05).

T1: (Basal diet (BD); control); T2:(BD + PBLM 2 g/kg); T3:(BD + PBLM 4 g/kg); T4:(BD + PBLM 8 g/kg); T5: (BD + POLM 2 g/kg); T6: (BD + POLM 4 g/kg); T7: (BD + POLM 8 g/kg).

^!Data were analysed as a 3 × 2 factorial arrangement, excluding control group.

The main effects data of second-growth phase of broiler chickens showed that as supplements dose increased, VH of duodenum and jejunum significantly (P < 0.05) increased, however, CD and VH: CD of duodenum and jejunum were not affected by increased dose of supplements. Furthermore, increased supplementation dose did not influence the VH, CD and VH: CD of ileum. A significant interaction was observed for duodenum VH (P < 0.0001), CD (P < 0.008) and VH: CD (P < 0.001), as well as, for jejunum VH (P < 0.0001), also for ileum CD (P < 0.006), and VH: CD (P < 0.05), thus, indicating that increased supplement concentration positively modulate the gut. On day 42, except for dietary group T₄ the increased (P < 0.05) VH were observed for duodenum and jejunum of all supplemented treatments in comparison with the control. Furthermore, significantly maximum (P < 0.05) VH were observed in T₃ and T₇, while recorded least (P < 0.05) VH in T₄ among treatments. Additionally, the VH of ileum was not significantly influenced (P > 0.05) by dietary inclusion of PBLM and POLM in experimental chickens. The CD of the duodenum was recorded increased (P < 0.05) in T_1, T₄ and T₅ as compared to other treatments T₃ and T₇; additionally the CD of ileum was recorded significantly lesser (P < 0.05) for T₃ and T₇. While the supplementation of PBLM and POLM has not affected the CD of jejunum. The dietary supplementation of PBLM and POLM resulted in higher (P < 0.05) VH: CD of duodenum and ileum in diets T₃ and T₇ relative to other treated and the control group. However, VH: CD of jejunum was recorded higher (P < 0.05) in T₃ relative to the control group. The current findings endorsed the idea, that phytogenic feed additives (PFAs) or phytobiotics favourably modify the gut structure and digestive functions, thus enhanced the growth in broiler chickens. The morphological changes of gut under the influence of phytobiotics may provide the information on their beneficial role in the digestive tract. Generally, phytobiotics have potential to increased VH across the gut, decrease inflammatory events, and improve absorption efficiency (Murugesan et al., 2014). Endorsing this idea, Khan et al. (2017) in a study observed that the inclusion of Moringa oleifera at a rate of 1.2% in the diet of broiler chickens positively modulate the gut health through a developed intestinal microarchitecture. In another study, Karangiya et al. (2016) observed that supplementation of garlic in broilers at a dose rate of 1%, resulted in higher mean villi length and width. The significant factors of gut health are the height of villi and crypt depths. Longer villi resulted in bigger surface area, that increase absorption of nutrients and thus enhanced the growth performance and health of chickens (Kamboh et al., 2015). The improved villi length is related to a better expression of brush border enzymes and enhanced transport of nutrients (Viveros et al., 2011). In this study, the increased villus height, recorded in dietary groups T₃ and T₄, that could be an explanation for enhanced growth and better FCR obtained at supplementation level of PBLM 4 g/kg and POLM 8 g/kg.

Since intestinal crypts serve as a source of epithelial cells for villus, and their depth is directly related to epithelial cell turnover. Phytobiotics or PAFs supplementation resulted in shallow crypts of the gut, that indicates less cellular turnover and healthy intestine. It is worth mentioning that the process of cellular turnover is an energy-utilizing event; if this energy utilisation is saved by reducing cellular turnover might be used for enhancement in growth events. Thus, longer villi and shallow crypts are indicative of better productive performance (Ahsan et al., 2018).

In the present study, it was noted that the increasing concentration of PBLM 8 g/ kg (T₄) resulted in reduced VH and increased CD, the current results corroborated the findings of Oso et al. (2019), who observed that inclusion of phytogenic blend that contained Piper betle in broiler chickens ration resulted in an increased duodenal VH at 1%, however decreased at a higher inclusion level of 2%; as well as this high concentration of phytogenic blend resulted in deepest crypt in jejunum; this suggested that the gut morphology improved at 1% inclusion beyond that the concentration get worsen.

The histological photomicrograph of duodenum and jejunum villi of broiler chickens on day 42 from all groups is presented in (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7).

Fig. 1.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from Control group T₁ (H & E) 100X.

Fig. 2.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from Control group T₂ (H & E) 100X.

Fig. 3.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from Control group T₃ (H & E) 100X.

Fig. 4.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from Control group T₄ (H & E) 100X.

Fig. 5.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from supplemented group T₅ (H & E) 100X.

Fig. 6.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from supplemented group T₆ (H & E) 100X.

Fig. 7.

Photomicrograph of histology of Duodenum (D) and Jejunum (J) villi of broiler chickens from supplemented group T₇ (H & E) 100X.

3.3. Nutrient digestibility

Supplementation of phytobiotics has been resulted in better feed intake and its utilisation, as they can enhance digestive enzymes concentrations; that promote nutrients digestion and increased absorption in the intestine (Khan et al., 2012, Abudabos et al., 2017). The results of apparent ileal nutrients digestibility (AID) are presented in (Tables 5 and 6). The main data effects of both growth phases of boiler chickens designated that DM, EE, CP and ash were not affected by the increased dose of supplementation, furthermore, no significant interaction was recorded. On day 21 and 42, the AID of dry matter (DM) and ether extract (EE) were observed higher (P < 0.05) in T₃ and T₇ among supplemented groups and relative to the control group. Furthermore, higher (P < 0.05) digestibility of crude protein (CP) was recorded in T₇ followed by T₃, T₂, and T₆ compared to dietary treatment T₅ and the control group; however, significantly least (P < 0.05) CP value was noted in T₄. This reduced digestion of crude protein CP in broilers fed on diet T₄ might be due to the high concentration of tannins, that adversely influenced on nutrient digestion, especially protein digestion (Sav́on et al., 2006). The results of the current study endorsed the findings of Mansoori et al. (2015), who revealed that 0.2% supplementation of chestnut tannin in the broilers decreased animal growth performance, by minimising the nutrient digestion and absorption. Very much similar results narrated by Farahat et al. (2017) who revealed that the dietary supplementation of grape seed extract tannin in broiler chickens at the concentration of 2% positively promoted the digestion of the nutrients, while, further increment in level negatively influenced the protein and amino acid digestion. On day 21 higher (P < 0.05) value of ash was noted in supplemented treatments relative to the control; however, ash was unaffected by supplemented diets on day 42. Current study outcomes are in line with Murugesan et al. (2015), who reported that the growth enhancement of broiler birds fed PFAs, might be associated with the increased digestibility of nutrients. Similarly, Hassan et al. (2015) described that supplementation of R. officinalis, S. Officinalis, and D.vulgaris in broilers ration resulted in increased digestibility of DM, CP, ether extract, and organic material. Generally, flavonoids and polyphenols improve cellular and mucosal immunity by regulating the circulatory and endocrine markers; thus, dietary supplementation of phytobiotics containing secondary metabolite like flavonoids can improve the immunity and health of broiler chickens (Kamboh et al., 2018).

Table 5.

Apparent ileal nutrients digestibility of broilers supplemented with different levels of PBLM and POLM on day 21.

Treatments	Dry matter (DM)	Ether extract (EE)	Crude protein (CP)	Ash
T1	69.34b	64.97b	68.20b	30.97b
T2	72.08b	68.28b	70.94 a	32.12 a
T3	76.15 a	74.03 a	74.02 a	33.92 a
T4	70.70b	65.85b	65.63c	32.51 a
T5	71.15b	66.19b	68.30b	32.17 a
T6	71.17b	68.07b	70.83 a	32.07 a
T7	75.30 a	72.69 a	74.17 a	33.31 a
SEM	1.33	1.33	1.37	0.46
Main effects!
Supplement concertation
2 g / kg	71.61	67.24	69.62	32.15
4 g / kg	73.66	71.05	72.48	32.99
8 g / kg	73.00	69.27	69.90	32.91
Supplements
P. betle	72.98	69.39	70.50	32.85
P. odorata	72.54	68.98	70.84	32.52
Source of variation
Supplement dose	0.845	0.578	0.697	0.775
Supplements	0.882	0.892	0.911	0.758
Supplement × Supplement dose	0.418	0.209	0.165	0.582

^abcThe means with different superscripts within the same columns are significantly different (P < 0.05).

T1: (Basal diet (BD); control); T2:(BD + PBLM 2 g/kg); T3:(BD + PBLM 4 g/kg); T4:(BD + PBLM 8 g/kg); T5: (BD + POLM 2 g/kg); T6: (BD + POLM 4 g/kg); T7: (BD + POLM 8 g/kg).

^!Data were analysed as a 3 × 2 factorial arrangement, excluding control group.

Table 6.

Apparent ileal nutrient s digestibility of broilers supplemented with different levels of PBLM and POLM on day 42.

Treatments	Dry matter (DM)	Ether extract (EE)	Crude protein (CP)	Ash
T1	71.84b	67.81b	69.71 bc	32.9
T2	74.37b	70.94b	73.47 a	34.71
T3	77.23 a	76.11 a	75.01 a	34.89
T4	73.04b	67.63b	67.88c	34.21
T5	73.01b	68.14b	71.02b	34.57
T6	73.07b	70.14b	72.06b	34.28
T7	78.06 a	74.07 a	75.31 a	34.74
SEM	1.18	1.3	1.35	0.44
Main effects!
Supplement concertation
2 g / kg	73.69	69.54	72.25	34.64
4 g / kg	75.15	73.13	73.69	34.73
8 g / kg	75.55	70.85	71.60	34.47
Supplements
P. betle	74.88	71.56	72.22	34.70
P. odorata	74.71	70.78	72.80	34.53
Source of variation
Supplement dose	0.83	0.60	0.85	0.98
Supplements	0.95	0.79	0.85	0.86
Supplement × Supplement dose	0.37	0.21	0.29	0.84

^abcThe means with different superscripts within the same columns are significantly different (P < 0.05).

^{T1: (Basal diet (BD); control); T2:(BD+ PBLM 2g/kg); T3:(BD+ PBLM 4g/kg); T4:(BD+ PBLM 8g/kg); T5: (BD+ POLM 2g/kg); T6: (BD+ POLM 4g/kg); T7: (BD+ POLM 8g/kg).}

^!Data were analysed as a 3 × 2 factorial arrangement, excluding control group.

4. Conclusions

The findings of the current study revealed that the dietary supplementation with Piper betle leaf meal and Persicaria odorata leaf meal enhanced the biological production of broiler chickens. The broiler chickens fed on dietary supplementation PBLM 4 g/ kg and POLM 8 g/kg of feed getting maximum BWG, positive modulation of gut structure and attained superior feed efficiency. Based on the results of the current study, the dietary supplementation of PBLM 4 g/kg and POLM 8 g/kg would be the appropriate doses and could be the potential candidates as natural alternative growth promoters in poultry production. Further studies are warranted to explore the comparative efficacy of PBLM and POLM with AGPs in broilers ration.

5. Authors’ contributions

MAB, AAK, LTC, SAA, and AS contributed in the study design and carried out statistical analysis. MAB involved in feeding trial. MAB, AAK, UK, and SBI conducted sample collection. MAB, AAK and UK performed laboratory analysis. MAB, AAK, LTC, SAA, AS, UK, and SBI contributed to the preparation of the manuscript. The final manuscript was reviewed and approved by all authors.