Synergistic effect of probiotic, chicory root powder and coriander seed powder on growth performance, antioxidant activity and gut health of broiler chickens.

Gut health plays an important role on production and performance of broilers. This trial was undertaken with an aim to evaluate the synergistic effect of probiotic, chicory root powder and coriander seed powder on the performance and gut health of broiler chicken. For this purpose, a total of 240 day-old broiler chicks were randomly allotted to six dietary treatments with 8 replicates of 5 birds in each. Treatment groups included T1 as control i.e., basal diet (BD) without any growth promoter and T2-BD + antibiotic (BMD 0.05%). In the remaining experimental diets, T3-probiotic (@ 0.01%) + chicory root powder (@ 1.0%), T4-probiotic (@ 0.01%) + coriander seed powder (@ 1.5%), T5-chicory root powder (@ 1.0%) + coriander seed powder (@ 1.5%) and T6-probiotic (@ 0.01%) + chicory root powder (@ 1.0%) + coriander seed powder (@ 1.5%). The results indicated that supplementation of probiotic + chicory (T3), probiotic + coriander (T4), chicory + coriander (T5) and probiotic + chicory + coriander (T6) in combination resulted in significantly (P<0.05) higher weight gain and better FCR compared to control and antibiotic groups at 42 d of age. Supplementation of different dietary groups did not show any significant (P>0.05) effect on feed intake of broilers. Supplementation of all the test diets (T3 to T6) significantly (P<0.05) increased the glutathione peroxidase (GSHPx), glutathione reductase (GSHRx) and superoxide dismutase (SOD) enzyme activity when compared to control and antibiotic groups at 42 d of age. Supplementation of all the test diets (T3 to T6) significantly (P<0.05) lowered the pH in the gut, increased Lactobacillus counts, and reduced E. coli and Salmonella counts in the ileum compared to control and antibiotic groups. Supplementation of all the test diets (T3 to T6) significantly (P<0.05) increased the villus height (VH), crypt depth (CD), VH:CD ratio and villus width (VW) in the duodenum and only VH and CD in the ileum compared to control and antibiotic groups. Significantly (P<0.05) higher jejunal VH and VW and increased the goblet cell number in duodenum, jejunum and ileum was recorded in all test diets (T3 to T6) compared to control and antibiotic groups. Therefore, combinations of probiotic (0.01%), chicory root powder (1.0%) and coriander seed powder (1.5%) can be used as feed additive for improving performance and gut health of broiler chicken.

Introduction

Antibiotics are being used as growth promoters in the poultry diets all over the world. However, in recent years, there has been rising demand to reduce or abolish the use of antibiotics as growth promoters due to the detrimental human health issue of antibiotic resistance [1]. Consumers’ awareness of poultry products that do not contain antibiotic residues has increased, encouraging the use of suitable alternatives to antimicrobial compounds [2]. Among the feed additives, probiotics, prebiotics, organic acids, enzymes and medicinal plants have drawn more attention due to their prophylactic and growth promoting effects. Thus, the use of probiotics, medicinal plants, herbs and spices in poultry diets has become more popular worldwide as an alternative to antibiotics to minimize the disease incidence and achieving better performance in chicken [2].

Probiotics are single or mixed cultures of live microorganisms which beneficially affect the host by improving the balance of intestinal flora [3]. Probiotics maintain the beneficial intestinal microflora by competitive exclusion and antagonism, lowering the gut pH through acid fermentation, limiting the damage caused by pathogenic bacteria [4], improving epithelial cell integrity (villi height and width), producing bacteriocins, stimulating the gut associated immune system and increasing the production of short-chain fatty acids. Recently, herbal feed additive products like chicory root powder are gaining attention as they indirectly promote antimicrobial action by reducing the harmful bacteria in the gut. Dried chicory root powder is a good source of inulin type fructans and oligofructose chains known for having prebiotic action without any toxicity [5]. Inulin-type fructans are indigestible carbohydrates, recognized as dietary fibers that improve intestinal health and bird’s performance through their prebiotic properties [6]. 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 [7, 8]. Similarly, probiotic + prebiotic supplementation decreased intestinal pH and viscosity in broilers [9]. Addition of probiotics (Lactobacillus acidophilus and lactose fermenting enterobacteria) and prebiotic combinations in broilers significantly (P<0.05) increased the villus height and crypt depth of the duodenum, ileum and jejunum [10].

Coriander (Coriandrum sativum) is regarded as both herb and spice, and has been used in medicine for thousands of years. Coriander seeds possess antioxidant, diuretic, anti-diabetic, hypocholesterolemic, antimicrobial, anthelmintic and anti-mutagenic qualities [11, 12]. Coriander seed powder contains 0.5–1.0% essential oil (carvone, geraniol, limonene, borneol, camphor, elemol and linalool) having antimicrobial properties against food borne pathogen such as Salmonella species [13]. In addition, it has appetizing and stimulatory effects in the digestion process by increasing production of digestive enzymes and juices, which stimulates digestion and peristaltic motion, thus improves feed efficiency [14, 15]. Coriander seed powder as an alternative to antibiotic growth promoter has been recommended for feeding in broilers by several authors [16-18]. In view of the above, this experiment was designed to evaluate the synergistic effect of probiotic (0.01%), chicory root powder (1.0%) and coriander seed powder (1.5%) on the performance, antioxidant status and gut health of broiler chicken.

Materials and methods

To conduct the study, two hundred and forty (240) day old commercial (Vencobb 400) broiler chicks were procured, individually weighed and wing banded. The birds were distributed randomly into 6 dietary treatments, each with 8 replicates having 5 chicks in each replicate. The chicks were reared in battery brooders under standard managemental conditions. The experimental period was from day old to 42 d of 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). All the treatment concentrations in the feed were weight/weight basis. Treatment groups (Table 2) included T1 as control i.e., basal diet (BD) without any growth promoter and T2—BD + antibiotic (Bacitracin Methylene Disalicylate at 0.05%–manufacturer Zoetis). In the remaining experimental diets, T3—probiotic (@ 0.01%) + chicory root powder (@ 1.0%), T4—probiotic (@ 0.01%) + coriander seed powder (@ 1.5%), T5—chicory root powder (@ 1.0%) + coriander seed powder (@ 1.5%) and T6—probiotic (@ 0.01%) + chicory root powder (@ 1.0%) + coriander seed powder (@ 1.5%). The probiotic contains 109 CFU/g of lyophilized and microencapsulated Bacillus coagulans, saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Bacillus subtilis, Enterococcus faecium, Bifidobacterium bifidum. The chemical composition of chicory root powder and coriander seed powder was given in Table 3. The dose levels (Probiotic at 0.01%, chicory at 1.0% and coriander at 1.5% levels) were selected based on previous trail results conducted.

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	55.9	56.4	60.0
Oil	2.10	4.0	5.0
Soyabean meal (CP 46%)	37.1	34.8	30.1
Stone grit	1.58	1.83	1.88
Dicalcium phosphate	1.85	1.90	1.96
Salt (NaCl)	0.45	0.49	0.49
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
Choline chloride (50%)	0.15	0.15	0.15
Toxin binder	0.10	0.10	-
Total	100	100	100
Nutrient composition (calculated values)
ME (kcal/kg)	2964	3075	3167
Crude protein (%)	22.90	21.65	19.65
Lysine (%)	1.28	1.21	1.10
Methionine (%)	0.53	0.49	0.47
Calcium (%)	0.95	1.04	1.06
Available phosphorous (%)	0.45	0.45	0.45

*Trace mineral provided per kg diet (Avimin): Manganese 80mg, Zinc 70g, Iron 40g, Copper 8mg, Iodine 1gm and Selenium 0.25g.

**Vitamin AB2D3K each gram contains (Nicomix): Vitamin A 82500IU, Vitamin D3 12000IU, Vitamin B2 50mg, Vitamin K 10mg

*** Vitamin B-Complex each gram contains (Nicomix); Vitamin B1 4mg, Vitamin B6 8mg, Vitamin B12 40mcg, Niacin 60mg, Calcium Pantothenate 40mg, Vitamin E 40mg.

Table 2

Experimental diets.

Trt	Experimental Diets
T1	Basal diet (BD) without additive
T2	BD + Antibiotic (BMD @ 0.05%)
T3	BD + Probiotic (@ 0.01%) + Chicory root powder (@ 1.0%)
T4	BD + Probiotic (@ 0.01%) + Coriander seed powder (@ 1.5%)
T5	BD + Chicory root powder (@ 1.0%) + Coriander seed powder (@ 1.5%)
T6	BD + Probiotic (@ 0.01%) + Chicory root powder (@ 1.0%) + Coriander seed powder (@ 1.5%)

Table 3

Chemical composition of Chicory root powder.

Composition (%)	Chicory root powder	Coriander seed powder
Moisture	3.16	3.23
Crude protein	14.55	14.91
Fat	1.76	0.96
Ash	3.98	8.88
Crude fiber	30.01	34.21
Total carbohydrates	48.76	50.12
Inulin	46.89	-

Brooder temperature was maintained at 34 ± 1°C up to 7 days of age and then gradually reduced to 26 ± 1°C by 21 days of age after which chicks were maintained uniformly at room temperature. Feed and water were offered ad libitum throughout the experimental period. Weekly body weight, Feed intake and feed conversion ratios were calculated as feed intake per unit bodyweight gain at weekly intervals. The mortality rate was recorded throughout the experiment. The metabolic trial was conducted with one bird from each replicate to determine the retention efficiency of Dry Matter (DM), Crude Protein (CP) and energy as per the procedures described by AOAC (1997) [19]. The antioxidant enzymes such as glutathione Peroxidase (GSHPx), glutathione Reductase (GSHRx) and superoxide Dismutase (SOD) were estimated by following the methods of Paglia and Valentine (1967) [20], Carlberg and Mannervik (1985) [21] and Madesh and Balsubramanian (1998) [22] respectively.

Before slaughter, the birds were fasted overnight with free access to water and sacrificed by cervical dislocation and allowed for complete bleeding for 5 to 7 minutes. 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 [23].

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 [24]. 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 eight 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 cellSens 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.

Record of temperature was maintained on daily basis where the highest daily average temperature recorded is 39.15°C and the lowest temperature is 20.8°C during the experimental period. The average relative humidity is 68.65 during the experimental period. The experiment was conducted during February and march- 2020.

Data analyzed for mean, standard errors and analysis of variance as per method of [25] and comparison of means were done [26] 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. No consent was raised by animal ethics committee while obtaining permission.

Results and discussion

Body weight gain

The results clearly indicated that supplementation of all test diets (T3 to T6) exhibited significantly (P<0.05) higher body weight gain compared to control (T1) and antibiotic (T2) groups at 42 days of age. The highest cumulative body weight gain (2185g) was recorded in probiotic + chicory (T3) combination group followed by probiotic + coriander (T4), chicory + coriander (T5) and probiotic + chicory + coriander (T6) groups. However, the lowest weight gain was recorded in control (T1) and antibiotic (T2) groups (Table 4). These results are in line with the findings of Taherpour et al. (2009) [27], who reported supplementation of probiotic and prebiotic combination improved the final body weight of broilers at 42 d of age. Similarly, supplementation of probiotic + prebiotic and probiotic + enzyme combination increased the body weight of broilers compared to control at 42 d of age [9]. Barad et al. (2017) [17] observed higher body weight gain in coriander seeds supplemented group when compared to control, turmeric powder and black pepper groups. Contrary to above results, Hofacre et al. (2003) [28] and Al-Khalaifa et al. (2019) [29] did not find positive effect on body weight in broilers fed with prebiotic + probiotic combination at 28 d of age.

Table 4

Synergistic effect of probiotic, chicory root powder and coriander 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	1975d	3533	1.79c
T 2	Antibiotic	2016c	3566	1.69bc
T 3	Probiotic + Chicory	2185a	3553	1.63a
T 4	Probiotic + Coriander	2149b	3564	1.66ab
T 5	Chicory + Coriander	2144b	3561	1.66ab
T 6	Probiotic + Chicory + Coriander	2140b	3545	1.65ab
SEM		10.627	15.869	0.0082
N		8	8	8
p-value		0.001	0.113	0.001

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

The highest mean weight gain was recorded in probiotic + chicory (T3) combination group which was significantly (P<0.05) higher among all the treatments. Similarly, Sanja et al. (2015) [30] reported addition of synbiotics (Enterococcus faecim + fructooligosaccharides) improved the body weight of broilers. Supplementation of probiotics and inulin combinations significantly (P <0.05) improved body weight gain in broilers [31]. The complimentary effect of probiotic and chicory powder on cumulative body weight gain as observed in the present study might be due to the suppression of undesirable microorganisms that lead to improved health status [32], increased nutrient digestibility, greater nutrient retention and improved gut health [33]. Similarly, increased body weight gains upon feeding diets containing probiotic + prebiotic combination [34-36] and probiotic + herb combination [37] in broiler chicken. Significant reduction in the counts of E. coli and Salmonella and reduction in gut pH by the supplementation of probiotic + chicory, probiotic + coriander, chicory + coriander and probiotic + chicory + coriander combinations in the present study is also in support with the authors. Contrary to above results, supplementation of probiotic + prebiotic combination did effect on body weight gain in broilers [10, 38, 39]

Feed Intake (FI)

The ANOVA revealed that there were no significant (P>0.05) differences in feed intake among different dietary treatments during overall experimental period (Table 4). The feed intake values at 42 d of age ranged between 3533 g to 3566 g. Similarly, supplementation of probiotic + prebiotic combination did not have significant (P>0.05) on FI of broilers [29]. In agreement with the results of this study, a series of earlier studies demonstrated that addition of probiotics + prebiotics [9, 36], chicory root powder [40, 41] and coriander seed powder [42] to the diet did not result in significant (P>0.05) effect on feed intake of broilers. On the contrary, probiotics + prebiotics [30], probiotic + herb combination [37], chicory root powder [30, 43] and coriander seed powder [17, 18] to the diets resulted in significant (P<0.05) effect on feed intake of broilers. These variations may be due to environmental factors and levels of the additives used in the experiment.

Feed conversion ratio (feed intake/ body weight gain)

Supplementation of probiotic + chicory (T3), probiotic + coriander (T4), chicory + coriander (T5) and probiotic + chicory + coriander (T6) combination groups significantly (P<0.05) improved the efficiency of feed utilization compared to control and antibiotic. However, broilers fed with the probiotic + chicory (T3) combination group was more efficient at converting feed to body mass during entire experimental period (Table 4). To stimulate the growth of beneficial bacteria in the gut using a probiotic + chicory (T3) combination was more effective than the other combinations in this study. This might be due to symbiotic relation between chicory inulin and probiotic. Chicory root powder inulin serves as a source of nutrient for the probiotic bacterial cultures for early establishing in the gut. Similar results were reported by Szakacs et al. (2015) [44] and Sanja et al. (2015) [30] who stated that probiotic + prebiotic combination improved feed efficiency in broilers. Ashayerizadeh et al. (2009) [45] reported that addition of antibiotic, probiotic + prebiotic combination improved FCR compared to control. Improved feed efficiency with probiotic 0.4% + prebiotic 0.2% was also reported by Utami and Wahyono (2019) [36]. In agreement with the results of probiotic + coriander combination in this experiment, Hedayati and Manafi (2018) [37] reported that probiotic and herbal compound supplementation significantly (P<0.05) improved feed conversion ratio compared to control and antibiotic in broilers. However, in contrary to our findings, Kirkpinar et al. (2018) [9] and Al-Khalaifa et al. (2019) [29] did not find positive effect of probiotic and prebiotic combination on FCR of broilers.

Improvement in feed conversion efficiency in treatment groups might be attributed to enhanced digestive enzymes activity and an encouraged growth of the beneficial micro-flora in the GIT induced by dietary supplementation of probiotic, chicory root powder and coriander seed powder combination [46, 47]. Mode of action of above feed additives differs from one another, but in general they are all considered as antimicrobial agents. Improvement in feed efficiency might be obtained by several factors like alteration in intestinal pH, suppression of growth of intestinal pathogens, enhancement of growth of non-pathogenic bacteria and improvement of intestinal function (increased villi height, crypt depth and integrity) and nutrient digestibility.

Nutrient utilization

Supplementation of all the test diets (T3 to T6) significantly (P<0.05) improved the energy retention, protein utilisation and dry matter digestibility compared to antibiotic, control groups (Table 5). The increased nutrient utilization in treatment groups might be due to probiotic bacteria, prebiotic properties of inulin and essential oils in coriander seed powder. Inulin-type fructan is a soluble fermentable fiber that is not digested by host digestive enzymes and serves as a substrate for beneficial like bifidobacteriae and lactobacilli in the lower part of the intestinal tract, the caeca and colon must be considered the sites of their effects on reabsorption of nutrients [48]. The mechanism by which inulin-type fructans may stimulate absorption is not well-known. The hypothesis more accepted is that the fermentation of inulin type fructans in the large intestine results in the production of short-chain fatty acids and lowers the gut pH. A lower intestinal pH facilitates absorption of nutrients [49, 50]. Similarly, Yang et al. (2008) [51] reported that supplementation of mannanoligosaccharides improved the energy and protein utilization in broilers. Justina et al. (2018) [52] indicated that supplementation of β-mannanase in broilers improved the dry matter digestibility and nutrient utilization. The enhanced dry matter digestibility and nutrient utilization may be attributed to the essential oils in coriander seed powder, which not only act as antibacterial and antioxidant, but also as stimulant of digestive enzymes in the intestinal mucosa, which might have improved the utilization of nutrients [15]. Similar results were also reported by Barad et al. (2017) [17] and Reddy et al. (2019) [53].

Table 5

Synergistic effect of probiotic, chicory root powder and coriander powder on nutrient utilization of broiler chicken.

Trt	Diets	Energy %	Protein %	Dry matter %
T 1	Control	70.52c	80.11c	72.65c
T 2	Antibiotic	72.25b	82.06b	74.18b
T 3	Probiotic + Chicory	75.77a	84.94a	76.89a
T 4	Probiotic + Coriander	75.52a	84.16a	76.80a
T 5	Chicory + Coriander	75.21a	84.11a	76.02a
T 6	Probiotic + Chicory + Coriander	75.68a	84.01a	76.92a
SEM		0.510	0.611	0.402
N		8	8	8
p-value		0.001	0.002	0.002

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

Antioxidant enzyme activity

The glutathione peroxidase (Units/ml) enzyme activity was significantly (P<0.05) higher with all the test diets (T3 to T6) compared to control (T1) and antibiotic (T2), the highest enzyme activity being recorded in probiotic + chicory combination (T3) and probiotic + coriander combination (T4) groups. The other groups showed intermediate glutathione peroxidase enzyme activity. However, Supplementation of all test diet (T3 to T6) significantly (P<0.05) increased the glutathione reductase and superoxide dismutase enzyme activity compared to control and antibiotic (Table 6). Increased concentration of antioxidant enzymes in our study, is an indicator of better free radical scavenging of test diets. The steady state of antioxidant enzymes activity in all test groups may reflect a significant improvement in health and oxidative status of the birds. In agreement with the results, Tagang et al. (2013) [54] and Shen et al. (2014) [55] recorded increased (P<0.05) activity of serum catalase and glutathione peroxidase enzymes with probiotics in broilers. Similar results were also reported by Dong et al. (2019) [56] and Tengfei et al. (2019) [57] with probiotic supplementation in broilers.

Table 6

Synergistic effect of probiotic, chicory root powder and coriander powder on antioxidant enzyme activity of broiler chicken at 42 d of age.

Trt	Diets	Glutathione peroxidase (Units/ml)	Glutathione reductase (Units/ml)	Superoxide dismutase (Units/mg protein)
T 1	Control	243d	1636b	6.62b
T 2	Antibiotic	314c	1664b	7.01ab
T 3	Probiotic + Chicory	397a	1782a	7.37a
T 4	Probiotic + Coriander	393a	1792a	7.25a
T 5	Chicory + Coriander	363b	1814a	7.24a
T 6	Probiotic + Chicory + Coriander	365b	1835a	7.39a
SEM		8.042	15.67	0.069
N		8	8	8
P-value		0.001	0.001	0.005

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

In agreement with the positive results of chicory root powder on antioxidant activity, Sanja et al. (2015) [30] reported that addition of synbiotics (Enterococcus faecim + fructooligosaccharides) significantly (P<0.05) increased serum glutathione peroxidase, peroxidase, glutathione reductase and catalase enzyme activities compared to the control group. Similarly, Wang et al. (2018) [58] observed increased total antioxidant capacity in prebiotics than control and antibiotic (Aureomycin) in broilers. Decreased lipid peroxidation levels and increased activity of the superoxide dismutase and catalase enzymes in broilers fed with inulin was reported by Andreia et al. (2020) [59].

The increase in activity of these antioxidant enzymes with supplementation of probiotics and chicory root powder might be due to better control of intestinal pathogens in the gut. Aerobic bacteria (Bacillus spp) use oxygen in the intestine to provide an anaerobic environment for the colonization of anaerobic bacteria, such as Lactobacilli and Bifidobacteria. Therefore, these lactic acid-producing bacteria produce a more acidic environment, which impairs the growth of opportunistic pathogens [60]. Lactobacillus acidophilus increased the hydroxyl radical and hydrogen peroxide scavenging ability. Lactic acid bacteria could produce certain factors to capture reactive oxygen species (ROS) and prohibit the cytotoxic activity of ROS [61]. Significant reduction in the gut pH, E. coli and Salmonella count and increased in Lactobacilli count in all test diets also supported by the authors. Contrary to these findings, probiotics [54, 55], chicory powder [30] did not have any positive effect on antioxidant enzyme activity in broilers.

Chitra and Leelamma (1999) [11] demonstrated that coriander had a better antioxidative effect by increasing the activity of glutathione peroxidase, glutathione reductase and superoxide dismutase enzyme compared to control. Coriander is an egregious source of phyto-chemicals and functional compounds namely polyphenols, flavonoids and ascorbic acid which ultimately constitute for its high antioxidant activity. Darughe et al. (2012) [62] demonstrated that essential oil of coriander contains camphor, cyclohexanol acetate, limonene, α-pinene and inhibited the rate of primary and secondary oxidation products formation and their effects were almost equal to BHA. The improvement in the antioxidant enzyme activity observed with the addition of coriander seed powder could be attributed to the presence of essential oils and their main components, linalool, trepene and terpenoid [63].

Gut pH

Supplementation of all test diets (T3 to T6) significantly (P<0.05) lowered the pH in duodenum, jejunum, ileum and caecum (except proventriculus) compared to control and antibiotic groups (Table 7). Probiotic bacteria produce short chain acids like lactic, acetic and other organic acids, which are responsible for reduction in the intestinal pH [64]. Aerobic bacteria (Bacillus spp) use oxygen in the intestine to provide an anaerobic environment for the colonization of anaerobic bacteria, such as Lactobacilli and Bifidobacteria. Therefore, these lactic acid-producing bacteria produce a more acidic environment, which impairs the growth of opportunistic pathogens [60]. Significant increase in the counts of Lactobacilli in test diets also support the above results. Al-Khalaifa et al. (2019) [29] reported that supplementation of probiotics and prebiotics driven the gut pH value towards acidity, but it failed to reach significance, as it was only a numerical difference. Whereas, Denli et al. (2003) [65] reported that inclusion of probiotic at 0.1% and antibiotic at 0.15% in broiler diets did not have any effect on the intestinal pH.

Table 7

Synergistic effect of probiotic, chicory root powder and coriander powder on gut pH of broiler chicken at 42 d of age.

Trt	Diets	Proventriculus	Duodenum	Jejunum	Ileum	Caecum
T1	Control	3.54	6.11c	6.43d	6.66c	7.39c
T2	Antibiotic	3.56	6.01b	6.30c	6.45b	7.18b
T3	Probiotic + Chicory	3.51	5.89a	6.06a	6.23a	6.94a
T4	Probiotic + Coriander	3.48	5.88a	6.15ab	6.30a	6.95a
T5	Chicory + Coriander	3.54	5.94ab	6.20b	6.31a	6.99a
T6	Probiotic + Chicory + Coriander	3.54	5.94ab	6.18b	6.25a	7.03a
SEM		0.0098	0.0155	0.0208	0.0270	0.0301
N		8	8	8	8	8
p-value		0.157	0.001	0.001	0.001	0.001

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

Supplementation of all the test diets (T3 to T6) including antibiotic group significantly (P<0.05) decreased the E. coli counts compared to control (Table 8). The lowest E. coli counts were recorded in antibiotic group (T2), probiotic + chicory (T3) and probiotic + chicory + coriander (T6) groups followed by probiotic + coriander (T4) and chicory + coriander (T5) groups. Supplementation of antibiotic (T2) significantly (P<0.05) decreased the ileal Salmonella counts compared to control and other test diets. The Salmonella count in probiotic + chicory + coriander (T6), probiotic + coriander (T4) groups and probiotic + chicory (T3) showed intermediate values, but they had significantly (P<0.05) lower Salmonella counts than the control (T1) and chicory + coriander (T5) groups. In agreement with the above results, Karwan et al. (2016) [31] observed that addition of postbiotics and inulin combinations significantly (P<0.05) reduced the Enterobacteriaceae count compared to control. Similarly, Biswas et al. (2018) [8] reported that supplementation of antibiotics (BMD) and prebiotics (MOS and FOS) reduced the total anaerobes and coliforms counts in ileum of broilers. Supplementation of antibiotic, probiotic and herbal compound significantly (P<0.05) reduced the E. coli, Salmonella and coliforms counts compared to control in broilers was reported by Hedayati and Manafi (2018) [37]. Probiotic bacteria produce short chain acids which decreases the intestinal pH and encourages the growth of Lactobacilli and Bifidobacteria [64]. Therefore, these lactic acid-producing bacteria produce a more acidic environment, which impairs the growth of opportunistic pathogens [60]. Significant increase in Lactobacilli count in all test diets was also support the above results. On contrary, Wang et al. (2018) [58] did not find any significant (P>0.05) difference in total anaerobic bacterial count in broilers with probiotic and antibiotic supplementation.

Table 8

Synergistic effect of probiotic, chicory root powder and coriander powder on gut microbiota (log10 of cfu/g count) in ileum sample of broiler chicken.

Trt	Diets	Escherichia coli (log10 cfu/g) *	Salmonellea spp. (log10 cfu/g) **	Lactobacillus spp. (log10 cfu/g) *
T 1	Control	8.29d	4.23d	7.89c
T 2	Antibiotic	7.15a	3.23a	6.93d
T 3	Probiotic + Chicory	7.18a	3.92c	8.08a
T 4	Probiotic + Coriander	7.33b	3.90c	7.86c
T 5	Chicory + Coriander	7.42c	4.16d	7.88c
T 6	Probiotic + Chicory + Coriander	7.19a	3.81b	7.94b
SEM		0.059	0.049	0.055
N		8	8	8
P-value		0.001	0.001	0.001

Values 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).

In agreement with the lowered E. coli and Salmonella counts in probiotic + coriander (T4), chicory + coriander (T5) groups, Ghazanfari et al. (2015) [66] reported that supplementation of antibiotic and coriander oil lowered the caecal E. coli counts than control in broilers. Similarly, Taha et al. (2019) [18] observed that coriander seed powder decreased the total bacterial, E. coli and C. perfringens counts in the ileum of broilers. The decreased pathogenic bacterial load in the ileum might be due to essential oils in coriander have which hydrophobic properties [67] that affect cell wall lipids of the bacteria by disturbing bacterial structures and rendering them more permeable, thus results in lower number of harmful bacteria.

The Lactobacilli counts were significantly (P<0.05) increased with the supplementation of probiotic + chicory (T3) followed by probiotic + chicory + coriander (T6) when compared to other treatment groups. Birds supplemented with antibiotic in the diet showed significantly (P<0.05) lower Lactobacilli counts. However, no significant (P>0.05) difference was recorded among control (T1), probiotic + coriander (T4) and chicory + coriander (T5) groups. Similarly, Dong et al. (2019) [56] reported that supplementation of microencapsulated probiotics significantly (P<0.05) increased the Lactobacilli counts in caecum of broilers. Similarly, Biswas et al. (2018) reported that supplementation of antibiotics (BMD) and prebiotics (MOS and FOS) increased the Lactobacilli counts in ileum of broilers. Increased faecal lactic acid bacteria in postbiotics and inulin combination groups was also reported by Karwan et al. (2016) [31] in broilers. Wang et al. (2018) [58] observed that microencapsulated probiotics and prebiotics (MEP) significantly (P<0.05) increased the Lactobacilli counts than control and antibiotic in broilers. In contrary, supplementation of probiotic did not have significant (P<0.05) effect in cecal Lactobacilli counts of broilers [29].

The increased Lactobacilli count in probiotic + chicory (T3) combination group might be due to probiotic bacteria such as Lactobacillus spp. or Bifidobacterium spp. use inulin for fermentation more efficiently than other groups of bacteria and produces short chain fatty acids on inulin to create an acidic environment which suppresses the growth of acid intolerant bacteria like Salmonellae and E. coli and enhanced the growth of acid tolerant bacteria like Lactobacilli and Bifidobacterium [68].

Gut histomorphometry

Supplementation of all the test diets (T3 to T6) significantly (P<0.05) increased the villus height (VH), crypt depth (CD), VH:CD ratio and villus width (VW) in the duodenum and only VH and CD in the ileum compared to control and antibiotic groups. Significantly (P<0.05) higher jejunal VH and VW was recorded in all test diets compared to control and antibiotic groups. Increased villus height and villus width enhances the absorptive capacity of the small intestine by reducing the digesta passage rate and therefore, optimize broiler performance. However, supplementation of different dietary groups did not show any significant (P>0.05) effect on jejunal CD and VH:CD ratio and ileal villus width at 42 d of age (Tables 9–11 and Figs 1–12).

Table 9

Synergistic effect of probiotic, chicory root powder and coriander powder on histomorphometry of duodenum of broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)
T 1	Control	1025.84c	240.58c	4.29c	134.11c
T 2	Antibiotic	1174.89b	260.43c	4.55b	156.05bc
T 3	Probiotic + Chicory	1598.52a	315.58b	5.15a	213.64a
T 4	Probiotic + Coriander	1261.81b	308.45b	4.10d	171.78b
T 5	Chicory + Coriander	1623.28a	319.91ab	5.07a	168.11b
T 6	Probiotic + Chicory + Coriander	1651.62a	343.38a	4.87ab	199.69a
SEM		43.420	6.825	0.087	5.764
N		8	8	8	8
P-value		0.001	0.001	0.001	0.001

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

Table 11

Synergistic effect of probiotic, chicory root powder and coriander powder on histomorphometry of ileum of broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)
T 1	Control	562.15d	172.87b	3.28c	131.15
T 2	Antibiotic	668.51c	158.67b	4.17b	129.05
T 3	Probiotic + Chicory	758.38b	160.25b	4.63b	136.76
T 4	Probiotic + Coriander	1019.99a	199.06a	5.22a	145.61
T 5	Chicory + Coriander	752.38b	166.24b	4.47b	131.87
T 6	Probiotic + Chicory + Coriander	742.35b	193.22a	3.83c	134.18
SEM		25.265	4.294	0.116	2.246
N		8	8	8	8
P-value		0.001	0.001	0.001	0.336

Values 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).

H&E, 2x.

Fig 12

Photomicrograph of the cross section of ileum from probiotic + chicory + coriander group (T6), H&E, 2x.

Photomicrograph of the cross section of Jejunum from antibiotic group (T2).

Photomicrograph of the cross section of ileum from control group (T1).

Photomicrograph of the cross section of ileum from antibiotic group (T2).

In agreement with the above results, Karwan et al. (2016) [31, 69] reported that addition of probiotics and inulin combinations increased the villus height and crypt depth of the duodenum, ileum and jejunum compared to control. Similarly, supplementation of probiotic + organic acid combination significantly (P<0.05) increased the duodenal VH, CD and VH:CD in broilers [70]. The positive effect of probiotics and 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 [71]. Probiotics and chicory root powder increase to production of short chain fatty acids and reduce intestinal pH. Hence, beneficial effects on intestinal tissue health and morphology are achieved. In contrary, Fernandes et al. (2014) [10] reported that supplementation of probiotic + prebiotic combination did not have any significant (P>0.05) effect on intestinal integrity of broiler.

Probiotic, chicory root powder and coriander seed 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 increases secretory function, digestion, and absorption of nutrients [8]. It is hypothesised that the increase in beneficial microbial activity resulting from dietary probiotic, chicory root powder and coriander seed powder supplementation may influence gut morphology and consequently affect gut maturation.

Conclusion

Supplementation of probiotic + chicory, probiotic + coriander, chicory + coriander and probiotic + chicory + coriander combinations produced greater weight gain, improved FCR, and higher antioxidant activity compared to control and antibiotic at 42 d of age. The combination of probiotic (0.01%) with chicory root powder (1.0%) was more effective than combinations of other additives in terms of body weight gain and FCR. Supplementation of different combinations of probiotic, chicory root powder and coriander seed powder significantly lowered the gut pH, E. coli and Salmonella counts and increased the Lactobacilli counts. In addition, this treatment improved the gut morphometry parameters such as VH, CD and VW in the in the intestines. Thus, supplementation of probiotic at 0.01%, chicory root powder at 1.0%, and coriander seed powder at 1.5% combinations could be used in the diet as a potential growth promoter in broiler chickens. However, follow up large-scale studies under field conditions are necessary before recommending the compounds in the broiler diet.

(XLSX)

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7 Mar 2022

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Reviewer #1: Summary: Interesting work. Please carefully review my comments and prepare a point-by-point response for the same. Revise the manuscript where appropriate. Thanks.

TITLE: Replace chicken with chickens.

Abstract:

Line 32: typo, please correct. Remove the extra “to”

Line 33: Please represent probiotic supplementation as percentage similar to chicory powder.

Line 37: IF you are representing antibiotic addition as gm/ton, please represent probiotic the same way. To avoid this confusion, I recommend representing your supplementations as percentage for all treatments.

Line 37: Please double check the abbreviation of gram. I believe it is “g”.

Introduction:

LINE 83: Remove the addition sign and replace with and.

Material and methods:

Line 108-113: Please include a table to describe the treatments clearly.

Line 116: Are the total bacterial load 32 billion CFU/100 g or each probiotic was at this concentration? Is this a commercial product or designed by the authors? How did the authors check the viability of each bacteria in the probiotic cocktail?

Line 120-122: Why are the authors presenting results in this section? I would recommend to move all the results presentation in the result section.

Line 118: Please represent probiotic levels as percentage too.

Line 130: “Feed intake”, please change to lower case F.

Line 134: Please include more details on how enzyme activity was determined. Describe how samples were stored, processed, followed by what type of assay was used.

Line 138: Please explain clearly how the 240 birds were sampled. It appears that only 1 bird/replicate that is 5 birds/treatment were processed for pH, intestinal contents etc. What about the other birds in the replicates? Why could the authors not process all birds for all samples to increase statistical power.

Question: How many trials/studies were conducted with 240 birds (6 dietary treatments, 8 replicates)?

Question: Where have the authors described the methodology for nutrient utilization? The results for nutrient utilization have been described in table 4 but I could not find the methodology portion for this section.

Question: How did the authors selectively enrich for Salmonella from the samples? What enrichment procedure was employed to facilitate recovery of Salmonella? Did the authors conduct a Salmonella load test at the start of the trials before the treatments began?

Results and Discussion:

Question: Why did the author not run groups for probiotic, chicory and coriander alone in the trials. Without these treatments, it is difficult to discern which component (probiotic, chicory, coriander) is effective in modulating body weight gains, FCR etc.

Table 7: Please share how the statistical analysis for comparing log CFU counts was conducted. I am interested to review log CFU/g values along with SE for each treatment. Some of the values for Lactobacillus (T5 7.88 vs T6 7.94), Salmonella (T4 7.33 vs T5 7.42) are very close to be statistically different from each other.

Reviewer #2: The authors investigated the effect of incorporating a combination of probiotics, chicory root powder and coriander seed powder on broiler growth performance, gut health and antioxidant activity. This study is relevant to the poultry industry for utilizing alternatives to antibiotic growth promoters.

However, my review for the manuscript was done partially because I felt that the experimental design and the number of birds used for the respective time points lacked clarity. It was hard to make an assessment on the data sets provided in the study. In addition, description provided for the statistical analysis and the inferences derived seemed incomplete. I would be happy to provide my further review and comments on the manuscript after the authors have provided a clarity on the experimental design and the N value for respective end points used in the study.

I have provided some of my comments for the manuscript below.

Abstract

Line 32-33 – please rephrase sentence as appropriate indicating the aim/background of the study. The details of the experimental design can be incorporated in the second sentence.

Introduction

Line76-77 – the connotation for antimicrobial properties of chicory root powder cannot be directly attributed but rather should be indicated as an indirect effect. Kindly rephrase the sentence appropriately. Or rather, if the authors had planned to indicate the antimicrobial activity other potential constituents, that may also be explained here.

Line 84 – the information regarding postbiotics may be removed from here. The authors can alternately provide additional references that cite the effect of probiotics + inulin combinations on poultry gut health. Specifically, the authors may cite the strains of probiotics that have been used by other researchers in a concise manner.

Line 90-92 – It would be better if the compounds listed in brackets are briefly expanded and described in terms of their chemical and functional properties/antimicrobial activities.

Materials and Methods

Line 116 – Does this mean the concentration of each bacterial strain or is this the total lactic acid bacterial counts in general?

Line 116 and Table 1 – Isn’t this analysis termed Proximate Analysis?

Line 118-121 – The way in which the treatment groups used for selecting the ideal formulation was not clear. What were the treatment groups involved in the broiler trials to determine the ideal composition of the mix that was eventually used for the final study?

Line 132 – clearly specify the N value for the metabolic trial.

Line 155-170 – how were the Salmonella colonies confirmed just by observed the colony morphology from the SS agar. What if the colonies observed in the plates were from Proteus?

Line 171 – was a blinded histopathological evaluation and analysis performed for the respective tissue samples?

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15 Mar 2022

Response to Reviewers

Editorial comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Reply: Followed the journal style

2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

Reply: One of the publications was published by me, so there might be a chance of matching words as the methodology followed was the same. Duplicate reference in the reference section was identified and removed.

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Reply: Corrected the manuscript for language usage, spelling, and grammar.

4. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

Reply: Included in materials and methods

5. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

Reply: Raw data was included

6. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

Reply: Raw data was provided. Kindly update Data Availability statement.

Reviewer #1:

1. TITLE: Replace chicken with chickens.

Reply by author: Modified as chickens

2. Line 32: typo, please correct. Remove the extra “to”

Reply:Corrected

3. Line 33: Please represent probiotic supplementation as percentage similar to chicory powder.

Reply: Modified as per reviewer suggestion

4. Line 37: IF you are representing antibiotic addition as gm/ton, please represent probiotic the same way. To avoid this confusion, I recommend representing your supplementations as percentage for all treatments.

Reply:Expressed all additives in percentage

5. Line 37: Please double check the abbreviation of gram. I believe it is “g”.

Reply: Modified as ‘g’

Introduction:

LINE 83: Remove the addition sign and replace with and.

Reply: Removed typographical error

Material and methods:

Line 108-113: Please include a table to describe the treatments clearly.

-Included

Line 116: Are the total bacterial load 32 billion CFU/100 g or each probiotic was at this concentration? Is this a commercial product or designed by the authors? How did the authors check the viability of each bacteria in the probiotic cocktail?

Reply: The probiotic used is a multistrain probiotic trail product produced by Intron Biologicals, Hyderabad and it contains a total of 109 CFU/g of lyophilized and microencapsulated Bacillus coagulans, Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Bacillus subtilis, Enterococcus faecium and Bifidobacterium bifidum. The viability was checked at the production itself.

Line 120-122: Why are the authors presenting results in this section? I would recommend to move all the results presentation in the result section.

Reply: Those lines were modified according to the materials and methods section. Removed lines 117 to 123.

Line 118: Please represent probiotic levels as percentage too.

Reply: Modified

Line 130: “Feed intake”, please change to lower case F.

Reply: Corrected

Line 134: Please include more details on how enzyme activity was determined. Describe how samples were stored, processed, followed by what type of assay was used.

Reply: *Complete procedure of antoxidant enzyme activity has given below.

*ANTIOXIDANT RESPONSES

The antioxidant enzymes such as glutathione Peroxidase (GSHPx), glutathione Reductase (GSHRx) and superoxide Dismutase (SOD) were estimated by following the methods of Paglia and Valentine (1967), Carlberg and Mannervik (1985) and Madesh and Balsubramanian (1998) respectively.

3.8.1 Glutathione Peroxidase (GSHPx) Enzyme Activity in Serum

GSHPx activity was determined by the method proposed by Paglia and Valentine (1967) with slight modifications. Microtiter plates (96 well) were used to measure Glutathione peroxidase activity. To the 12.5 μl of serum, 250 μl of 0.1mM PBS (pH 7.4), 12.5 μl of H2O2 and 12.5 μl of reduced glutathione were added to wells and incubated at room temperature for 5 minutes, following which 12.5 μl of nicotinamide adenine dinucleotide phosphate (NADPH) solution was added and optical density was measured at 340 nm against the blank using ELISA reader - μQuant (BioTek instruments) for 5 minutes at 60 seconds interval and expressed as units/mg protein.

Glutathione Reductase (GSHRx) Enzyme Activity in Serum

GSHRx activity was determined according to method described by Carlberg and Mannervik (1985) with slight modifications. Microtiter plates (96 well) were used to measure Glutathione reductase activity. To the 12.5 μl of serum, 250 μl of 0.1mM PBS (pH 7.4), 12.5 μl of oxidized glutathione, 12.5 μl of FAD and12.5 μl of 80 mM EDTA were added and incubated at room temperature for 15 minutes. Optical density was measured at 340 nm against the blank by using ELISA reader - μQuant (BioTek instruments) for 5 times at 60 seconds interval after addition of 12.5 μl of NADPH solution at last and expressed as units/mg protein.

Superoxide Dismutase (SOD) Enzyme Assay in Serum

Microtiter plates (96 well) were used for assay of SOD activity. To the 100 μl of test sample, 6 μl of 1.25 mM 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was added in duplicate for each sample. 15 μl of 100 μM pyrogallol and 29 μl of 25 mM PBS were added to make the volume to 150 μl. Pyrogollol was freshly prepared and added after the addition of all other reagents and incubated for 10 minutes at room temperature and the reaction was terminated with addition of 150 μl of dimethyl sulfoxide (DMSO), which arrests the reaction and dissolves the MTT formazan crystals formed. Plates were shaken well and optical density recorded at 570 nm using ELISA reader - μQuant (Madesh & Balsubramanian, 1998).

Line 138: Please explain clearly how the 240 birds were sampled. It appears that only 1 bird/replicate that is 5 birds/treatment were processed for pH, intestinal contents etc. What about the other birds in the replicates? Why could the authors not process all birds for all samples to increase statistical power.

Reply: One bird from each replicate means 8 birds for each treatment and total 48 samples were taken. According to the recommendations of IAEC, the number of birds to be sacrificed was limited as n=8 is enough for statistical analysis.

Question: How many trials/studies were conducted with 240 birds (6 dietary treatments, 8 replicates)?

Ans: Only one trial was conducted with 240 birds.

Question: Where have the authors described the methodology for nutrient utilization? The results for nutrient utilization have been described in table 4 but I could not find the methodology portion for this section.

Ans: We did not mentioned complete procedure of metabolic trial in methodology, however, we followed the standard procedure recommended by AOAC (1997). The metabolic trial was conducted with one bird from each replicate to determine the retention efficiency of Dry Matter (DM), Crude Protein (CP) and energy as per the procedures described by AOAC (1997).

Question: How did the authors selectively enrich for Salmonella from the samples? What enrichment procedure was employed to facilitate recovery of Salmonella? Did the authors conduct a Salmonella load test at the start of the trials before the treatments began?

Ans: 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. Salmonella-Shigella agar (SS Agar) for Salmonella spp. was used.

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 minutes, 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 (Gunal et al., 2006). Aerobic bacterial plates ( Salmonella spp.) were placed in an incubator at 37oC 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.

We did not conduct salmonella load test at the start of experiment as it was not required for our experiment.

Results and Discussion:

Question: Why did the author not run groups for probiotic, chicory and coriander alone in the trials. Without these treatments, it is difficult to discern which component (probiotic, chicory, coriander) is effective in modulating body weight gains, FCR etc.

Ans: Sir, we have conducted three separate experiments (other than this) with probiotic @ 10 g, 15 g, and 20 gm per 100kg, coriander @ 0.5%, 1.0% and 1.5% & chicory root powder @ 0.5%, 1.0% and 1.5%. The results of above experiments clearly indicated that each group from probiotic, chicory root powder and coriander treatments had shown desirable performance at particular dosages. Probiotic at 10g/100 kg, chicory at 1.0 % and coriander at 1.5 % levels significantly (P<0.05) increased the overall performance of broilers when compared to control, antibiotic and other levels of respective treatments.

The above experimental results/data was already been published in Plosone and Indian journal of animal sciences.

I am sharing the link of above article for your reference.

https://doi.org/10.1371/journal.pone.0260923

Table 7: Please share how the statistical analysis for comparing log CFU counts was conducted. I am interested to review log CFU/g values along with SE for each treatment. Some of the values for Lactobacillus (T5 7.88 vs T6 7.94), Salmonella (T4 7.33 vs T5 7.42) are very close to be statistically different from each other.

Ans: mentioned below

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

Treatment Replicates colonies cfu/ml (106 dilution0 log 10 value

T1 1 192 192000000 8.28

1 201 201000000 8.30

1 195 195000000 8.29

1 189 189000000 8.28

1 199 199000000 8.30

1 188 188000000 8.27

1 195 195000000 8.29

1 200 200000000 8.30

T2 2 12 12000000 7.08

2 14 14000000 7.15

2 12 12000000 7.08

2 15 15000000 7.18

2 14 14000000 7.15

2 15 15000000 7.18

2 16 16000000 7.20

2 16 16000000 7.20

T3 3 18 18000000 7.26

3 16 16000000 7.20

3 15 15000000 7.18

3 15 15000000 7.18

3 12 12000000 7.08

3 14 14000000 7.15

3 15 15000000 7.18

3 16 16000000 7.20

T4 4 16 16000000 7.20

4 19 19000000 7.28

4 18 18000000 7.26

4 25 25000000 7.40

4 22 22000000 7.34

4 23 23000000 7.36

4 24 24000000 7.38

4 25 25000000 7.40

T5 5 18 18000000 7.26

5 32 32000000 7.51

5 28 28000000 7.45

5 29 29000000 7.46

5 26 26000000 7.41

5 28 28000000 7.45

5 27 27000000 7.43

5 25 25000000 7.40

T6 6 16 16000000 7.20

6 14 14000000 7.15

6 15 15000000 7.18

6 16 16000000 7.20

6 18 18000000 7.26

6 13 13000000 7.11

6 17 17000000 7.23

6 16 16000000 7.20

Reviewer #2:

- Abstract

Line 32-33 – please rephrase sentence as appropriate indicating the aim/background of the study. The details of the experimental design can be incorporated in the second sentence.

Reply: Included as per recommendations.

Introduction

Line76-77 – the connotation for antimicrobial properties of chicory root powder cannot be directly attributed but rather should be indicated as an indirect effect. Kindly rephrase the sentence appropriately. Or rather, if the authors had planned to indicate the antimicrobial activity other potential constituents, that may also be explained here.

Reply: Modified according to the recommendations

Line 84 – the information regarding postbiotics may be removed from here. The authors can alternately provide additional references that cite the effect of probiotics + inulin combinations on poultry gut health. Specifically, the authors may cite the strains of probiotics that have been used by other researchers in a concise manner.

Reply: Incorporated accordingly.

Line 90-92 – It would be better if the compounds listed in brackets are briefly expanded and described in terms of their chemical and functional properties/antimicrobial activities.

Reply: Sir, They were already proven compounds as indicated by reference, no need to discuss in detail.

Line 116 – Does this mean the concentration of each bacterial strain or is this the total lactic acid bacterial counts in general?

Reply: Total bacterial count (Bacillus coagulans, saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus plantarum, Streptococcus thermophilus, Bacillus subtilis, Enterococcus faecium, Bifidobacterium bifidum).

Line 116 and Table 1 – Isn’t this analysis termed Proximate Analysis?

Reply: Yes sir, proximate analysis.

Line 118-121 – The way in which the treatment groups used for selecting the ideal formulation was not clear. What were the treatment groups involved in the broiler trials to determine the ideal composition of the mix that was eventually used for the final study?

Reply: Sir, we have conducted three separate experiments (other than this) with probiotic @ 10 g, 15 g, and 20 gm per 100kg, coriander @ 0.5%, 1.0% and 1.5% & chicory root powder @ 0.5%, 1.0% and 1.5%. The results of above experiments clearly indicated that each group from probiotic, chicory root powder and coriander treatments had shown desirable performance at particular dosages. Probiotic at 10g/100 kg, chicory at 1.0 % and coriander at 1.5 % levels significantly (P<0.05) increased the overall performance of broilers when compared to control, antibiotic and other levels of respective treatments.

The above experimental results/data was already been published in Plosone and Indian journal of animal sciences.

I am sharing the links of above articles for your reference.

https://doi.org/10.1371/journal.pone.0260923

Line 132 – clearly specify the N value for the metabolic trial.

Reply: N value was already mentioned in table no 4.

The metabolic trial was conducted with one bird from each replicate means 8 birds treatment and total 48 samples were used for estimation of Dry Matter (DM), Crude Protein (CP) and energy.

Line 155-170 – how were the Salmonella colonies confirmed just by observed the colony morphology from the SS agar. What if the colonies observed in the plates were from Proteus?

Reply: Translucent colonies with black centers which are typical to Salmonella on SS agar were counted by using colony counter and the number of colonies was expressed as log10 value.

The complete procedure was explained below.

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) for E. coli growth, Salmonella-Shigella agar (SS Agar) for Salmonella spp. and MRS agar (De Man, Rogosa and Sharpe agar) for Lactobacilli spp growth was used.

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 minutes, 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 (Gunal et al., 2006). Aerobic bacterial plates (E. coli, Salmonella spp.) were placed in an incubator at 37oC for 24 hours. Anaerobic (Lactobacilli spp.) medium plates were placed in an anaerobic jar with an anaerobic gas pack system at 37oC 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.

Line 171 – was a blinded histopathological evaluation and analysis performed for the respective tissue samples?

Reply: Yes sir, the HP slides were analyzed blindly, except the control group, so as to have a reference point. We have taken 8 histopathological sections per treatment each from each bird.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

28 Apr 2022

2 May 2022

Synergistic effect of probiotic, chicory root powder and coriander seed powder on growth performance, antioxidant activity and gut health of broiler chicken

1. Please ensure that you refer to Table 1 in your text as, if accepted, production will need this reference to link the reader to the Table.

Response: Table 1 has been included in text and please provide reference link to Table 1.

2. Please amend the title either on the online submission form or in your manuscript so that they are identical.

Response: corrected

3. Please provide additional details regarding participant consent. In the Methods section, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Response: No consent was raised by animal ethics committee while obtaining permission. Moreover, the consent was not required for conducting experiments in Broilers. The same has been included in ethics statement.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

7 Jun 2022

Synergistic effect of probiotic, chicory root powder and coriander seed powder on growth performance, antioxidant activity and gut health of broiler chickens

PONE-D-21-40040R2

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16 Jun 2022

PONE-D-21-40040R2

Synergistic effect of probiotic, chicory root powder and coriander seed powder on growth performance, antioxidant activity and gut health of broiler chickens

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Table 10

Synergistic effect of probiotic, chicory root powder and corianderpowder on histomorphometry of jejunum of broiler chicken.

Trt	Diets	Villus height (μm)	Crypt depth (μm)	Villus height: Crypt depth Ratio	Villus width (μm)
T 1	Control	949.13c	171.25	5.68	153.23b
T 2	Antibiotic	1010.44b	168.96	5.99	164.00b
T 3	Probiotic + Chicory	1082.86a	165.90	6.69	210.44a
T 4	Probiotic + Coriander	1013.05b	171.70	5.93	197.99a
T 5	Chicory + Coriander	1044.34ab	167.88	6.26	204.35a
T 6	Probiotic + Chicory + Coriander	1061.79ab	175.81	6.09	207.68a
SEM		10.135	1.672	0.106	4.257
N		8	8	8	8
P-value		0.001	0.638	0.107	0.001

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