Supplementation of chicory root powder as an alternative to antibiotic growth promoter on gut pH, gut microflora and gut histomorphometery of male broilers.

The experiment was conducted to study the effect of chicory root powder on the gut performance of broilers. For this purpose, two hundred commercial male broiler chicks were randomly divided into 5 treatment groups with 8 replications of 5 birds each and reared in battery brooders up to 42 days of age. The experimental design consisted of; T1 basal diet (BD) without antibiotic, T2: BD + antibiotic (BMD at 500 gm/ton), T3: BD + chicory root powder (0.5%), T4: BD + chicory root powder (1.0%), T5: BD + chicory root powder (1.5%). The results revealed that supplementation of 1.0% chicory root powder recorded significantly (P<0.05) higher body weight gain, feed intake and better feed conversion ratio (FCR) compared to antibiotic, control and 0.5 & 1.5% chicory powder at 42 days of age. Supplementation of various levels of chicory root powder significantly (P<0.05) lowered (P<0.05) the pH in duodenum, jejunum, ileum and caecum compared to control. Supplementation of chicory root powder (0.5, 1.0 & 1.5%) significantly (P<0.05) decreased the E. coli and Salmonella counts and (P<0.05) increased the Lactobacilli counts in ileum when compared to control and antibiotic groups. Supplementation of chicory (1.0% and 1.5%) groups significantly (P<0.05) increased the villus height (VH), crypt depth (CD), VH:CD ratio and villus width (VW) in the duodenum, ileum and jejunum at 42 d of age. Supplementation of chicory 1.0% and 1.5% groups significantly (P<0.05) increased the goblet cell number in duodenum, jejunum and ileum of broilers. Therefore, chicory root powder (1.0 and 1.5%) can be used as an alternative to antibiotic for improving gut performance of broiler chicken.

Introduction

The mode of action of antibiotics is by suppressing the negative effects of pathogenic bacteria in the gastrointestinal tract and increasing nutrients absorption in intestines. But in recent years, due to negative human health issue of antibiotic resistance, there is an increasing pressure to reduce or eliminate the use of antibiotics as growth promoters [1]. The increased awareness among consumers for the poultry products without antibiotic residue encouraged the utilization of suitable alternatives for antibacterial compounds [2]. Recently, products like chicory root powder and coriander seed powder gaining attention as herbal feed additives having antimicrobial properties (Due to inulin). Chicory is a perennial herbaceous plant and their roots are baked, roasted, ground and used as coffee additive. Fresh chicory root contains 11–15% inulin and it may increase up to 40% in dried chicory root powder. Dried chicory root powder is a good source of inulin type fructans and oligofructose chains known for having prebiotic action without any toxicity [3]. Inulin-type fructans are indigestible carbohydrates, recognized as dietary fibers that improve intestinal health and bird’s performance through their prebiotic properties [4]. The fermentation activity of inulin inhibits the growth of harmful strains, selectively stimulates the growth of beneficial bacteria by decreasing the intestinal pH through increasing the absorption of short chain fatty acids and thus promotes the growth of broiler chickens [5, 6].

It has also been suggested that feeding chicory root powder to broiler can increase the absorption of nutrients by increasing jejunum villus height and crypt depth [7]. Similarly, probiotic + prebiotic supplementation decreased intestinal pH and viscosity in broilers [8]. Supplementation of prebiotics Increased the intestinal characteristics like villus total and villus height in broilers [9]. Addition of postbiotics and inulin combinations in broilers significantly (P<0.05) increased the villus height and crypt depth of the duodenum, ileum and jejunum [10]. In view of the above, this experiment was designed to evaluate the dietary supplementation chicory root powder as an alternative to antibiotic growth promoter on gut pH, gut ecology and morphometry of broilers.

Materials and methods

For this purpose, 200 day-old male broiler chicks (Vencobb) were distributed randomly in to five dietary treatments of eight replicates with five chicks in each replicate. At day one, chicks were wing banded and reared under optimum brooding conditions. The broilers were maintained in battery brooders with feed and water fed ad lib from 1 to 42 days age. The birds were fed with maize and soybean meal-based diets containing 2958, 3074 and 3163 kcal ME and 22.76, 21.58 and 19.68 percent crude protein, respectively during prestarter (0-14d), starter (15-28d) and finisher (28-42d) phases (Table 1). The experimental design consisted of; T1: Basal diet (BD) without antibiotic, T2: BD + antibiotic (Bacitracin Methylene Disalicylate at 500 gm/ton–manufacturer Zoetis), T3: BD + Chicory root powder (0.5%), T4: BD + Chicory root powder (1.0%), T5: BD + Chicory root powder (1.5%). The chemical composition of Chicory root powder was given in Table 2.

Table 1

Ingredient composition of basal diets (in kgs) fed to the commercial broilers from 0-42days.

Ingredient	Pre-starter (0-14d)	Starter (15-28d)	Finisher (29-42d)
Maize	56.09	56.4	59.8
Oil	2.04	4.0	5.0
Soyabean meal (CP 46%)	37.1	34.6	30.1
Stone grit	1.58	1.83	1.88
Dicalcium phosphate	1.85	1.90	1.96
Salt (NaCl)	0.46	0.46	0.48
DL-Methionine	0.22	0.18	0.16
L-Lysine HCl (99%)	0.17	0.15	0.13
Trace Mineral Mixture*	0.10	0.10	0.10
Vitamin AB2D3K**	0.020	0.020	0.020
Vitamin B-Complex**	0.025	0.025	0.025
Coccidiostat (Coxynil)	0.05	0.05	0.00
Choline chloride (50%)	0.15	0.15	0.15
Toxin binder	0.10	0.10	0.10
Total	100	100	100
Nutrient composition (calculated values)
ME (kcal/kg)	2958	3074	3163
Crude protein (%)	22.76	21.58	19.68
Lysine (%)	1.30	1.21	1.08
Methionine (%)	0.55	0.49	0.45
Calcium (%)	0.97	1.04	1.06
Available phosphorous (%)	0.45	0.45	0.45

*Trace mineral provided per kg diet: Manganese 120mg, Zinc 80mg, Iron 25mg, Copper 10mg, Iodine 1mg and Selenium 0.1mg.

**Vitamin premix provided per kg diet: Vitamin A 200000IU, Vitamin D3 12000IU, Vitamin E 10mg, Vitamin K 2mg, Riboflavin 25mg, Vitamin B1 1mg, Vitamin B6 2mg, Vitamin B12 40mg and Niacin 15mg.

Table 2

Chemical composition of chicory root powder.

Composition (%)	Chicory root powder
Moisture	3.16%
Crude protein	14.55
Fat	1.76
ash	3.98
Crude fiber	30.01
Total carbohydrates	48.76
Inulin	46.89

One bird from each replicate was sacrificed on 42nd day of age from each treatment group. Gut (proventriculus, gizzard, duodenum and ileum) pH was recorded immediately after collection of gastro-intestinal contents from respective part of gut. Approximately 1.0 g of sample content was suspended in 5ml distilled water, mixed vigorously with glass rod and pH was determined using digital pH meter. The electrode was rinsed with distilled water and recalibrated in between the readings [11].

Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 38.05°C and the lowest temperature is 18.5° C during the experimental period. The average relative humidity is 65.56 during the experimental period. The experiment was conducted during December and January—2020.

Gut ecology

Eight birds from each dietary treatment were slaughtered on 42nd day and intestines were dissected at Meckel’s diverticulum. Approximately 5g of ileal digesta was collected aseptically into sterile sampling tubes and immediately transferred on ice to the laboratory for microbiological examination for E. coli, Salmonella spp and Lactobacilli spp counts. Eosin methylene blue agar (EMB) was used for E. coli growth, Salmonella-Shigella agar (SS Agar) used for Salmonella spp. and MRS agar (De Man, Rogosa and Sharpe agar) used for Lactobacilli spp growth.

Then, 9 sterile test tubes with lids containing 9mL of phosphate buffer solution (PBS, pH-7.4) as diluent were prepared. Approximately 1g of the intestinal contents taken by sterile swab and homogenized for 3 min, aseptically mixed, added to the tubes, and diluted up to 109. Later, 1ml of the contents of each test tube was transferred to one of three selective agar media on petri plates, respectively [12]. Aerobic bacterial plates (E. coli, Salmonella spp) were placed in an incubator at 37°C for 24 hours. Anaerobic (Lactobacilli spp) medium plates were placed in an anaerobic jar with an anaerobic gas pack system at 37°C for 24 hours. Finally, the intestinal bacterial colony populations formed in each plate was counted by colony counter and the number of colonies was expressed as log10 value.

Histomorphometry

On 42nd day during slaughter, 2 cm long segment of duodenum, jejunum and ileum of six birds from each treatment were collected and then washed with physiological saline solution and fixed in 10% neutral buffered formalin solution. These samples were processed for histomorphological examination in terms of measurement of parameters like villous height (VH), cryptal depth (CD), villus width and villous height:crypt depth ratio. Histological technique involves processes like fixation of tissue, dehydration, clearing, embedding, cutting and staining. Fixation in 10% formalin with approximately 10–20 times the volume of the specimen was done. Tissues were dehydrated by using increasing strength of alcohol like 50%, 70%, 90% and 100%. Clearing was done by replacing alcohol by xylene for 0.5–1 hour. Impregnation of tissue with wax was done at melting point temperature of paraffin wax and the volume of wax was about 25–30 times the volume of tissues for a total duration of 4 hours. Impregnated tissues were placed in a mould with their labels and then fresh melted wax was poured in it and allowed to settle and solidify. These paraffin embedded tissues were sectioned at 5μm thickness and stained routinely with Hematoxylin-Eosin stain (H&E).

Histological sections were examined under 2X of light microscopy with micrometry and photographic attachment. The images were analyzed using image analyzing software (OLYMPUS cell Sens Standard, version 1.13). A total of 20 intact well oriented crypt-villous units per bird were selected randomly, measured and the mean length was calculated for each sample. Villous height was measured from the tip of the villi to the base between individual villi, and crypt depth measurements were taken from the valley between individual villi to the basal membrane.

Data analyzed for mean, standard errors and analysis of variance as per method of [13] and comparison of means were done [14] using software of Statistical Package for Social Sciences (SPSS) 20.0 version and significance was considered at P<0.05.

Ethical approval

All authors hereby declare that all biological trials have been examined and approved by the ethics committee of PV Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad, India (Institutional Animal Ethics Committee number: IV/2019-02/IAEC/CVSC, Hyderabad, India) and have therefore been performed in accordance with the ethical standards.

Results and discussion

Groups supplemented with CRP (1.0%) had significantly (P<0.05) higher weight gain compared to antibiotic, control and CRP (0.5 & 1.5%) treatment groups at 42 days of age (Table 3). Supplementation of 0.5% and 1.0% CRP significantly (P<0.05) increased the feed intake compared to antibiotic, control and 1.5% CRP groups. The best feed efficiency was recorded with CRP (1.0%) and antibiotic groups followed by CRP (0.5%) and CRP (1.5%), where as poor feed efficiency was noticed in control group at 42 d of age. The significant (P<0.05) improvement in body weight gain and feed efficiency by feeding the chicory root powder (1.0%) is in accordance with the earlier findings of Yusrizal and Chen (2003a) [15]; Karwan et al. (2016) [10]; Yousfi et al. (2017) [16] and Praveen et al. (2017) [17]. Supplementation of chicory root powder (1% and 3% levels) significantly (P<0.05) improved overall body weight gain than control [7]. Nabizadeh (2012) [18] stated that addition of inulin @ 1.0% level significantly (P<0.05) increased live body weight and feed efficiency of broilers at 42 d of age. However, contrary to our findings, Liu et al. (2011) [19] observed no significant (P>0.05) difference with supplementation of chicory root powder in broiler diets. It was hypothesized that the beneficial effect of chicory root powder in broilers might be due to prebiotic properties of inulin. Inulin-type fructan is a soluble fermentable fiber that is not digested by host digestive enzymes and serves as a substrate for beneficial bacteria in the gut of birds [20]. The fermentation activity inhibits the growth of Escherichia coli and Salmonella and selectively stimulates the growth of Bifidobacteria and Lactobacillus development in the gut [15].

Table 3

Effect of chicory root powder on body weight gain (g), feed intake and feed conversion ratio of broiler chicken.

Trt	Diets	Body weight gain	Feed intake	Feed conversion ratio
T 1	Control	2140c	3654bc	1.708c
T 2	Antibiotic	2214b	3677abc	1.663a
T 3	CRP (0.5%)	2202b	3711ab	1.684b
T 4	CRP (1.0%)	2263a	3729a	1.648a
T 5	CRP (1.5%)	2169c	3648c	1.683b
SEM		8.278	9.595	0.0042
N		8	8	8
p-value		0.001	0.020	0.001

Gut pH

pH values of various segments of the digestive system (except proventriculus) were significantly (P<0.05) influenced at the time of slaughter age (42 d). Supplementation of various levels of chicory root powder (0.5, 1.0 &1.5%) significantly (P<0.05) lowered the pH in duodenum, ileum, and caecum when compared to control and antibiotic groups (Table 4). However, supplementation of chicory (1.5%) group significantly (P<0.05) lower jejunum pH values compared to all other treatments. However, the pH values in chicory 0.5% and 1.0% supplemented groups were lower than the control and comparable with antibiotic. In agreement with the results of chicory root powder in this experiment, other researchers [18, 21] reported that supplementation of chicory inulin significantly (P<0.05) decreased the caecum pH in broilers compared to control. In contrast with present study, addition of chicory forage and root inclusion did not effect on caecal pH of broilers at 31 d of age [19]. The decreased intestinal pH might be due to beneficial intestinal microflora, such as Lactobacillus spp. or Bifidobacterium spp. They use inulin or oligofructose for fermentation to produce short chain fatty acids (acetate, propionate, butyrate and lactate) which creates an acidic environment in GIT of birds [15].

Table 4

Effect of chicory root powder on gut pH of broiler chicken at 42 d of age.

Trt	Diets	Proventriculus	Duodenum	Jejunum	Ileum	Caecum
T 1	Control	3.71	5.94c	6.63c	6.86c	7.30b
T 2	BMD	3.68	5.71b	6.44b	6.52b	7.21b
T3	CRP (0.5%)	3.68	5.41a	6.39b	6.40a	6.90a
T4	CRP (1.0%)	3.66	5.39a	6.38b	6.38a	6.83a
T5	CRP (1.5%)	3.70	5.45a	6.21a	6.31a	6.79a
SEM		0.031	0.038	0.025	0.034	0.038
N		8	8	8	8	8
p-value		0.988	0.001	0.001	0.001	0.001

Value bearing different superscripts within a column are significantly (P<0.05) different

Supplementation of antibiotic and chicory treatment groups significantly (P<0.05) decreased the E. coli and Salmonella counts compared to control at 42 d of age. The lowest E. coli counts were recorded in antibiotic group followed by chicory (1.0% & 1.5%) and chicory (0.5%) groups (Table 5). In agreement with the lowered E. coli and Salmonella counts in chicory groups [6], addition of prebiotics and antibiotics (BMD) lowered the total anaerobes and coliforms counts in broilers. Chicory root powder contains a fermentable fiber (Inulin) that is not digested by enzymes and serves as a substrate for beneficial bacteria in the gut. The fermentation activity inhibits the growth of E. coli and Salmonella and selectively stimulate the growth of Lactobacillus and Bifidobacteria in the gut [20]. Similarly, Yusrizal and Chen (2003a) [15] indicated that normal intestinal microflora, such as Lactobacillus spp. or Bifidobacterium spp., use chicory inulin for fermentation more efficiently than other groups of bacteria; these micro-organisms produce short chain fatty acids (SCFA) and lactate on inulin to create an acidic environment which suppresses the growth of E. coli and Salmonella. The reduction of pathogenic bacteria in the experiment may be due to the increased acidic pH in the intestines. On contrary, Wang et al. (2018) [22] reported prebiotics and antibiotic supplementation did not reduce caecal total anaerobic bacteria in broilers. Similarly, Nabizadeh (2012) [18] observed that inulin supplementation did not affect ileal E. coli counts in broilers. In agreement with the results of this study, a series of earlier studies demonstrated that addition chicory root powder [10, 21, 23] decreased the harmful bacterial count in broilers.

Table 5

Effect of chicory root powder on gut microbiota (log10 of cfu/g count) in ileum sample of broiler chicken.

Trt	Diets	Escherichia coli (log10 cfu/g) *	Salmonella spp (log10 cfu/g) **	Lactobacillus spp (log10 cfu/g) *
T 1	Control	8.10d	4.53d	7.75b
T 2	BMD	6.74a	3.27a	6.77c
T 3	CRP (0.5%)	7.07c	4.05c	7.79b
T 4	CRP (1.0%)	6.96b	3.91b	7.93a
T 5	CRP (1.5%)	6.92b	4.06c	7.89a
SEM		0.078	0.067	0.070
N		8	8	8
p-value		0.001	0.002	0.001

Value bearing different superscripts within a column are significantly (P<0.05) different

* Calculated as per log10 colony forming units/gram of sample (106).

** Calculated as per log10 colony forming units/gram of sample (103).

Supplementation of various levels chicory and control group significantly (P<0.05) increased the Lactobacilli counts in the ileum compared to antibiotic. The highest Lactobacilli counts were recorded in chicory 1.0% level and 1.5% level followed by chicory 0.5% and control groups. In agreement with the increased Lactobacilli counts in chicory groups, Yusrizal and Chen (2003a) [15] reported that addition of chicory fructans increased the Lactobacilli counts in the gizzard and small intestine contents of broilers. Similarly, Xu et al. (2003) [23] observed the enhanced growth of intestinal Bifidobacterium and Lactobacillus count with fructooligosaccharide supplementation in broilers. Corroborating the results of the present study, several researchers reported that addition of prebiotics increased the Lactobacillus counts in the ileum of broilers [5, 6, 22]. This might be due to inulin (fermentable fiber) in chicory root powder that is not digested by enzymes and serves as a substrate for beneficial bacteria in the gut. The fermentation activity inhibits the growth of E. coli & Salmonella and selectively stimulate the growth of Lactobacillus and Bifidobacteria in the gut [20]. On contrary, Nabizadeh (2012) [18] reported inulin supplementation had no effect on Bifidobacteria and Lactobacilli counts in ileum of broilers.

Gut histomorphometry

Supplementation of chicory (1.0%) level significantly (P<0.05) increased the duodenal villus height (VH), crypt depth (CD), VH:CD ratio and villus width (VW) when compared to control and antibiotic groups (Table 6). The duodenal morphometry parameters in chicory 0.5% and 1.5% groups were intermediate and was statistically comparable with antibiotic group. Significantly (P<0.05) higher jejunal VH and VH:CD ratio was recorded in chicory 1.0% group compared to chicory (0.5 and 1.5%), control and antibiotic groups (Table 7 and Figs 1–5). However, all chicory supplemented groups significantly (P<0.05) increased the jejunal VW compared to control and antibiotic groups. Supplementation of chicory (0.5, 1.0 and 1.5%) diets significantly (P<0.05) increased the ileal villi height (VH), crypt depth (CD) and VH:CD ratio in comparison with control and antibiotic groups (Table 8). Among all the treatments, the highest VH and CD was recorded in chicory (1.0%) group. Supplementation of chicory 1.0% and 1.5% groups significantly (P<0.05) increased the goblet cell number in duodenum, jejunum and ileum of broilers. chicory root powder may reduce the growth of many pathogenic and non-pathogenic intestinal bacteria thereby resulting in reduction in intestinal colonization and infectious process which ultimately decrease the inflammatory process of intestinal mucosa resulting in improved villus height and villus width which in turn improves digestive secretory function and absorption of nutrients [6]. It is hypothesised that the increase in beneficial microbial activity resulting from dietary chicory root powder supplementation may influence gut morphology and consequently affect gut maturation.

Table 6

Effect of chicory root powder on histomorphometry of duodenum in broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)	Goblet cell number
T 1	Control	1683.46b	216.99d	7.83a	225.00c	6.50d
T 2	BMD	1583.82c	260.37b	6.19b	338.21b	9.17c
T 3	CRP (0.5%)	1595.22c	265.26b	6.04b	378.94a	10.33b
T 4	CRP (1.0%)	1878.08a	306.77a	6.14b	377.93a	11.83a
T 5	CRP (1.5%)	1739.96b	242.54c	7.50a	217.57c	10.67b
SEM		21.767	5.606	0.160	13.624	0.374
N		6	6	6	6	6
p-value		0.001	0.001	0.001	0.001	0.001

Value bearing different superscripts within a column are significantly (P<0.05) different

Table 7

Effect of chicory root powder on histomorphometry of jejunum in broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)	Goblet cell number
T 1	Control	1202.51d	185.81	6.47b	183.01b	8.01c
T 2	BMD	1303.61c	192.48	6.78b	200.54b	10.67b
T 3	CRP (0.5%)	1546.11ab	215.62	7.19b	258.50a	11.83b
T 4	CRP (1.0%)	1626.77a	195.66	8.41a	252.78a	14.17a
T 5	CRP (1.5%)	1510.07b	208.24	7.41a	257.56a	13.67a
SEM		31.674	3.890	0.181	6.488	0.458
N		6	6	6	6	6
p-value		0.001	0.088	0.003	0.001	0.001

Value bearing different superscripts within a column are significantly (P<0.05) different

Fig 1

Photomicrograph of the cross section of jejunum from control group (T1), Hematoxylin-Eosin stain (H&E), 2x.

Fig 5

Photomicrograph of the cross section of jejunum from 1.5% chicory root powder group (T5), H&E, 2x.

Table 8

Effect of chicory root powder on histomorphometry of ileum in broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)	Goblet cell number
T 1	Control	863.77e	152.36c	5.82b	175.47	10.33c
T 2	BMD	1030.35d	207.91b	4.98d	181.18	13.83b
T 3	CRP (0.5%)	1151.16b	213.67b	5.39c	183.53	15.83a
T 4	CRP (1.0%)	1579.88a	239.14a	6.64a	180.63	17.50a
T 5	CRP (1.5%)	1095.46c	161.40c	6.91a	177.28	16.83a
SEM		44.631	6.526	0.142	1.810	0.533
N		6	6	6	6	6
p-value		0.001	0.001	0.001	0.668	0.001

Value bearing different superscripts within a column are significantly (P<0.05) different

In agreement with the increased gut morphology parameter in chicory supplemented groups, Yusrizal and Chen (2003a) [15] recorded increased jejunum villi distribution with inulin supplementation in broilers. Similarly, Xu et al. (2003) [23] reported increased ileal villus height, jejunal and ileal microvillus height and VH:CD ratios at the jejunum and ileum with fructooligosaccharide supplementation in broilers. Izadi et al. (2013) [7] observed that feeding chicory root powder (1% and 3%) to broilers significantly (P<0.05) increased the villus length, villus surface area, number of goblet cells and villus length/crypt depth ratio in the jejunum. Supplementation of MOS and FOS in broiler increased the intestinal crypt depth when compared to control and antibiotic groups was reported by Biswas et al. (2018) [6]. The positive effect of chicory root powder on the intestinal morphology mainly arose from its ability to create a favourable intestinal environment which had a better effect on intestinal morphology [23]. Prebiotics increase production of fatty acids and reduce intestinal pH. Hence, beneficial effects on intestinal tissue health and morphology are achieved. Corroborating the results of the present study, several researchers reported that addition of prebiotics increased the intestinal morphometry parameters of broilers [6, 9, 10, 18].

Supplementation of different dietary treatments did not affect jejunum CD and ileal VW at 42 d of age. Similarly, Ortiz et al. (2009) [24] and Rebole et al. (2010) [5] reported inulin supplementation had no effect on VH, CD and VW in the jejunum. Liu et al. (2011) [19] observed that chicory forage and root inclusion had no effect on jejunum morphology in broilers.

Conclusion

Finally, it can be concluded that supplementation of 1.0% chicory root powder was more effective in terms of higher weight gain, better FCR compared to other groups. Dietary supplementation of chicory root powder at 1.0% and 1.5% level significantly decreased the gut pH and reduced the harmful bacterial count in the intestines of broilers. And also increased the beneficial bacterial count and gut morphometry parameters like VH, CD, VW and goblet cell number in the small intestine. Hence, chicory at 1.0 and 1.5% level can be used as an alternative to antibiotic growth promoter in broiler chicken.

(XLSX)

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27 Jul 2021

PONE-D-21-14465

SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERS

PLOS ONE

Dear Dr. srinivas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

It is very important to research on animal sources of alternative plant sources and their derivatives to antibiotics that are banned for use as growth factors in poultry nutrition. However, the flawed experimental design and methodology greatly reduces the merits of this study.

# The introduction section are given the information about the choice of the treatments. Just a small general paragraph (“The increased awareness among consumers for the poultry products without antibiotic residue encouraged the utilization of suitable alternatives for antibacterial compounds”) informs that there are “antibacterial compounds” to be used. In the research, no antibacterial compound was used, but chicory root powder was used. Moreover, what are the biological active substances of this herb root powder? If only a experimental plan could be built on these compounds, the approach would be more accurate.

# According to which criteria the nutritional needs of the animal are determined, the animal's house environment humidity and temperatures, the litter used, etc. such as environmental factors could be given.

#Vitamin and mineral levels in the diet are given incorrectly.

# It is not clear whether the nutritional composition of the basal feed was calculated or analysed. Other feed additives should not be used in feed ingredients. In addition, the coccidiostat used in the diet is of antibiotic origin and cannot be used in finisher diets and is prohibited. Due to the danger of antibiotic residues in the tissues, the anticoccidiostat is removed from the diets of the broilers 7-15 days before slaughter.

# Since this research stands out as a preliminary experimental research, due to methodological deficiencies and flaws in terms of scientific quality.

# I would like to state that the criticisms of the referees came to the fore, especially with the fact that performance data should be given and the number of animals was insufficient.

# Ethics committee document should be provided.

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Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

It is very important to research on animal sources of alternative plant sources and their derivatives to antibiotics that are banned for use as growth factors in poultry nutrition. However, the flawed experimental design and methodology greatly reduces the merits of this study.

# The introduction section are given the information about the choice of the treatments. Just a small general paragraph (“The increased awareness among consumers for the poultry products without antibiotic residue encouraged the utilization of suitable alternatives for antibacterial compounds”) informs that there are “antibacterial compounds” to be used. In the research, no antibacterial compound was used, but chicory root powder was used. Moreover, what are the biological active substances of this herb root powder? If only a experimental plan could be built on these compounds, the approach would be more accurate.

# According to which criteria the nutritional needs of the animal are determined, the animal's house environment humidity and temperatures, the litter used, etc. such as environmental factors could be given.

#Vitamin and mineral levels in the diet are given incorrectly.

# It is not clear whether the nutritional composition of the basal feed was calculated or analysed. Other feed additives should not be used in feed ingredients. In addition, the coccidiostat used in the diet is of antibiotic origin and cannot be used in finisher diets and is prohibited. Due to the danger of antibiotic residues in the tissues, the anticoccidiostat is removed from the diets of the broilers 7-15 days before slaughter.

# Since this research stands out as a preliminary experimental research, due to methodological deficiencies and flaws in terms of scientific quality.

# I would like to state that the criticisms of the referees came to the fore, especially with the fact that performance data should be given and the number of animals was insufficient.

# Ethics committee document should be provided.

Thanks.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: in material and methods which requirements used to adjust feeding programme. in all feeding programm starter, grower and finisher if use prestarter used for 1st week and protein content 24%. so i see you can use starter(1-14) or follow the recommendation book of the chickens. in table of ingredients which type of Soya bean used(44-46-48). vitamin premix try to check its composition vitamin A may be 10000000 IU.

Nutrient composition calculated or analyzed?

Chicory root powder composition and its content of inulin and other essential oils or contents if possible.

BMD composition and concentration and manufacture.

digital pH meter which type its model?

in table 2 or table 3 use the same groups name Chicory (0.5%) or CRP (0.5%) and not use antibiotic may use BMD.

if possible insert dates about growth performance table and the effects of these Chicory root powder in feed intake and gain and FCR and other parameters to show effect of these treatments on broiler performance

Reviewer #2: Dear Authors Regarding the manuscript title SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERS

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

• Table 1 Ingredients and nutrient composition of the experimental diets, need to carful checking for the following :

1- SBM , add the crude protein level 44 or 46%?

2- MEn, kcal/kg, Ca, digP , not according to the Vencobb requirement.

• Introduction and Discussion

Need some other references about using antibiotic and its alternatives in broilers. I recommend you read the following references:

Ahmed A. Saleh, K. Amber and A. A. Mohammed (2020) Dietary supplementation with avilamycin and Lactobacillus acidophilus effects growth performance and the expression of growth-related genes in broilers. Animal Production Science. 60(14) 1704-1710

Ahmed A. Saleh , Tarek A. Ebeid , Alaeldein M. Abudabos. (2018) Effect of dietary phytogenics (herbal mixture) supplementation on growth performance, nutrient utilization, antioxidative properties and immune response in broilers. Environmental Science and Pollution Research. 25:14606–14613.

Ahmed. A. Saleh, Daichi Ijiri and Akira Ohtsuka (2014). Effects of summer shield supplementation on the growth performance, nutrient utilization, and plasma lipid profiles in broiler chickens. Journal of Veterinarni Medicina. 59, (11): 536–542.

• Results: it will be better if you present the data for performance in table.

Reviewer #3: Material and methods:

The author stated that in part of material and methods )One bird from each replicate was sacrificed on 42nd day of age from each treatment group. Gut (proventriculus, gizzard, duodenum and ileum) pH was recorded immediately after collection of gastro-intestinal contents from respective part of gut( .

The number of samples of one sample is very, very, very small. We cannot statistically analyze and rely on these results to generalize their results and be confident in their results. The number should not be less than three samples as a minimum.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: Yes: Hamada Ahmed

Reviewer #2: No

Reviewer #3: Yes: Lamiaa M. Radwan

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29 Sep 2021

the manuscript has been modified as per reviewers and editor suggestion (enclosed file)

SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERS

Reviewer comments Reply by author

The introduction section are given the information about the choice of the treatments. Just a small general paragraph (“The increased awareness among consumers for the poultry products without antibiotic residue encouraged the utilization of suitable alternatives for antibacterial compounds”) informs that there are “antibacterial compounds” to be used. In the research, no antibacterial compound was used, but chicory root powder was used. Moreover, what are the biological active substances of this herb root powder? If only a experimental plan could be built on these compounds, the approach would be more accurate. • The anti-microbial activity of chicory root powder due to inulin which contains prebiotic action

• Introduction was modified as per reviewer suggestion

• Biological active substance in chicory powder is - Inulin

According to which criteria the nutritional needs of the animal are determined, the animal's house environment humidity and temperatures, the litter used, etc. such as environmental factors could be given. • Temperature and humidity has been included in results.

• The experiment was conducted in battery brooders.

Vitamin and mineral levels in the diet are given incorrectly. Vitamin and mineral levels were added as per vencobb requiements.

It is not clear whether the nutritional composition of the basal feed was calculated or analysed. Other feed additives should not be used in feed ingredients.

In addition, the coccidiostat used in the diet is of antibiotic origin and cannot be used in finisher diets and is prohibited. Due to the danger of antibiotic residues in the tissues, the anticoccidiostat is removed from the diets of the broilers 7-15 days before slaughter. • nutritional composition of the basal feed was calculated values

• sir, we have used herbal origin coccidiostat that is coxynil

• moreover, we have withdrawn coccidiostats from finisher diets

Since this research stands out as a preliminary experimental research, due to methodological deficiencies and flaws in terms of scientific quality. • Chicory contains about 40 % inulin and inulin type fructans and oligofructose chains known for having prebiotic action and exert antimicrobial action. Based on this, the experiment was designed

I would like to state that the criticisms of the referees came to the fore, especially with the fact that performance data should be given and the number of animals was insufficient. • Sir, performance data is going to publish in other journal. That’s why it was not included in journal

Ethics committee document should be provided. • Sir, will be submitted during resubmission time

Reviewer #1:

Reviewer comments Reply by author

in material and methods which requirements used to adjust feeding programme. in all feeding programm starter, grower and finisher if use prestarter used for 1st week and protein content 24%. so i see you can use starter(1-14) or follow the recommendation book of the chickens.

in table of ingredients which type of Soya bean used(44-46-48). vitamin premix try to check its composition vitamin A may be 10000000 IU.

The birds were fed with maize and soybean meal-based diets containing 2958, 3074 and 3163 kcal ME and 22.76, 21.58 and 19.68 percent crude protein, respectively during prestarter (0-14d), starter (15-28d) and finisher (28-42d) phases. We followed the breeder recommendations of vencobb.

• Soya bean used was 46.

• Once agin, Vit pre mix composition was checked. It was 20000000IU only

Nutrient composition calculated or analyzed? Nutrient composition on Calculated values

Chicory root powder composition and its content of inulin and other essential oils or contents if possible. Sir, we have analyzed chicory root powder composition. Now, it has been included in Materials part

BMD composition and concentration and manufacture. Bacitracin Methylene Disalicylate Soluble Powder 50% is a granulated biomass premix containing 150 g (activity) of bacitracin methylene disalicylate per 1 kg of product. Bacitracin Methylene Disalicylate Soluble Powder 50%

Manufacture - Zoetis

digital pH meter which type its model? Labtronics LT-50 Table-Top Microprocessor Based pH meter

Model No.: LT-50

in table 2 or table 3 use the same groups name Chicory (0.5%) or CRP (0.5%) and not use antibiotic may use BMD.

Corrected as per reviewer suggestion

if possible insert dates about growth performance table and the effects of these Chicory root powder in feed intake and gain and FCR and other parameters to show effect of these treatments on broiler performance

Sorry sir, performance and other parameters are going to publish in other journal

Reviewer #2:

Reviewer comments Reply by author

- SBM , add the crude protein level 44 or 46%? Sir, 46 % level

2- MEn, kcal/kg, Ca, digP , not according to the Vencobb requirement. Feed requirements were calculated as per major and minor guides suggestion

Reviewer #3:

Reviewer comments Reply by author

The author stated that in part of material and methods.One bird from each replicate was sacrificed on 42nd day of age from each treatment group. Gut (proventriculus, gizzard, duodenum and ileum) pH was recorded immediately after collection of gastro-intestinal contents from respective part of gut( .

The number of samples of one sample is very, very, very small. We cannot statistically analyze and rely on these results to generalize their results and be confident in their results. The number should not be less than three samples as a minimum.

Sir, One bird from each replicate means – 8 samples per each treatment.

It is adequate and sufficient for statistical analysis

The figures of jejunum and Ileum were included in manuscript

The above manuscript has been modified as per reviewer suggestion

Submitted filename: Response to reviewers.docx

Click here for additional data file.

1 Nov 2021

PONE-D-21-14465R1SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERSPLOS ONE

Dear Dr. srinivas,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please review the referee comments again (about the completion of some methodological shortcomings, the correction of typographical errors, and the clear answer to general questions in terms of intelligibility to the readers) and make your final revision. Thank you

Please submit your revised manuscript by Dec 16 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

For your guidance, you can check the reviewers' comments..

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: in material and methods ad lib from write italic form

Soybean meal type (44-46% cp)- lysine concentration or pure grade

the amount of vitamin A and D in premix is low check the product again.

Coccidiostat which type chemical or ionophores

Chicory root powder proximate analysis (CP, EE, Ash.....) may affect chemical composition of the diet, please to correct total amount to adjust 100% of treated diets

Reviewer #2: Dear Authors Regarding the manuscript title SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERS

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, The authors answered all the inquiries and the manuscript may be accepted in this form

Reviewer #3: � He mentioned the following : Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 38.05 °C and the lowest temperature is 18.5 ° C during the experimental period. But the percentage of humidity has not been clarified, and it is known the importance of the relationship between temperature and humidity. Please clarify the degree of humidity during the experiment.

� Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 38.05 °C and the lowest temperature is 18.5 ° C during the experimental period. Please move this paragraph from the results and discussion section to the Material and methods.

� The data of body weights for the different treatments must be added in the manuscript to clarify the economic return of using supplementation of chicory root powder Is there a clear difference in weight when marketing?

Reviewer #4: ?Why were the growth rates and feed consumption rate for birds not calculated

?Salt means

.What is the percentage of protein in soybean meal? You can be written in the composition table No 1

It is preferable to mention the place of planting the plant, the time of harvest, the method of preservation and drying until analysis.

Reviewer #5: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Hamada Ahmed

Reviewer #2: No

Reviewer #3: Yes: Lamiaa M. Radwan (Poultry Production Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt )

Reviewer #4: No

Reviewer #5: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Submitted filename: Comment PONE-D-21-14465R1.docx

Click here for additional data file.

3 Nov 2021

Reviewer #1:

Reviewer comments Reply by author

in material and methods ad lib from write italic form

-------Changed to italic form

Soybean meal type (44-46% cp)- ---- Soyabean meal type has incorporated in Table no 2

lysine concentration or pure grade ----- Sir, Lysine is pure grade 99% and the same was incorporated in Table 2

the amount of vitamin A and D in premix is low check the product again.

--------Once again, Vit pre-mix composition was checked. Vit A is 20000000IU only and Vit D is 12000 IU

Coccidiostat which type chemical or ionophores ------We have used ‘coxynil’. It is a polyherbal preparation

Chicory root powder proximate analysis (CP, EE, Ash.....) may affect chemical composition of the diet, please to correct total amount to adjust 100% of treated diets ------- Feed requirements were calculated as per major and minor guides suggestion.

Though it was not mentioned, feed given to chicory supplemented groups was adjusted to basal diet requirements. i.e Prestarter (22.76) Starter (21.58) and Finisher (19.68)

Reviewer #2:

Reviewer comments Reply by author

- The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, The authors answered all the inquiries and the manuscript may be accepted in this form

-no comments from reviewer

Reviewer #3

Reviewer comments Reply by author

Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 38.05 °C and the lowest temperature is 18.5 ° C during the experimental period. But the percentage of humidity has not been clarified, and it is known the importance of the relationship between temperature and humidity. Please clarify the degree of humidity during the experiment.

----------Included relative humidity

Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 38.05 °C and the lowest temperature is 18.5 ° C during the experimental period. Please move this paragraph from the results and discussion section to the Material and methods. ---------------Moved to material and methods

The data of body weights for the different treatments must be added in the manuscript to clarify the economic return of using supplementation of chicory root powder Is there a clear difference in weight when marketing? Growth performance data has been included in results and discussion part

Reviewer 4

Reviewer comments Reply by author

Why were the growth rates and feed consumption rate for birds not calculated------ Growth performance data has been included in results and discussion part

Salt means ----- Salt means common salt (NaCl) and has included in table 2

What is the percentage of protein in soybean meal? You can be written in the composition table No 1 ------- 46 % CP soyabean meal was used and it has incorporated in feed composition table

It is preferable to mention the place of planting the plant, the time of harvest, the method of preservation and drying until analysis ------Pure Powdered chicory root was directly purchased from Ms Jyothi chicory, Aravind Industries, Guntakal – 515803, Andhra Pradesh, India

The data regarding the cultivation and harvest of Chicory is not available as we have not cultivated.

Reviewer 5

Reviewer comments Reply by author

• However, it is better if the author includes growth performance of the birds. The number of birds used in the experiment was also insufficient. -------- Growth performance data has been included in results and discussion part

• The author stated that one bird per replica was sacrificed which is very small and statistically to rely on. The minimum sample size for statistical analysis should be three. -------Sir, One bird from each replicate means – 8 samples per each treatment.

It is adequate and sufficient for statistical analysis (8 samples per treatment)

• Why the author focussed only on male broiler birds? Why you did not include female broiler birds? -------- male birds were selected to get uniform growth rate among the groups and to reduce error variation.

Growth performance data was added in results section and two references were newly added in reference section.

1. Ortiz LT, Rodri guez ML, Alzueta C, Rebole A, Trevin J. Effect of inulin on growth performance, intestinal tract sizes, mineral retention and tibial bone mineralization in broiler chickens. British Poultry Science 2009; 50 (3): 325-332.

2. Yousfi Z, Kazemi Fard M, Rezaei M, Ansari P Z. Effect of chicory extract and probiotic on performance, caracas characteristics, blood parameters, intestinal microflora and immune response of broiler chickens. 2017; (9): 185-195.

The above manuscript has been modified as per reviewer suggestion

22 Nov 2021

SUPPLEMENTATION OF CHICORY ROOT POWDER AS AN ALTERNATIVE TO ANTIBIOTIC GROWTH PROMOTER ON GUT pH, GUT MICROFLORA AND GUT HISTOMORPHOMETERY OF MALE BROILERS

PONE-D-21-14465R2

Dear Dr. srinivas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

Reviewer #5: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

Reviewer #5: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: (No Response)

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, The authors answered all the inquiries and the manuscript may be accepted in this form

Reviewer #3: (No Response)

Reviewer #4: Why was no analysis done of the bioactive components of the additive used in the experiment?

Why was the humidity level not made clear during the experiment? Including relative humidity

Why was an economic assessment such as the European production efficiency factor not done?

Reviewer #5: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #2: Yes: Prof. Ahmed Ali Saleh

Reviewer #3: Yes: Lamiaa M. Radwan (Poultry Production Department, Faculty of Agriculture, Ain Shams Univ., Cairo, Egypt )

Reviewer #4: Yes: Gamal Ali Abdelhafez Ali Hamady

Reviewer #5: No

1 Dec 2021

PONE-D-21-14465R2

Supplementation of chicory root powder as an alternative to antibiotic growth promoter on gut pH, gut microflora and gut histomorphometery of male broilers

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