Effects of an organic acids blend and coated essential oils on broiler growth performance, blood biochemical profile, gut health, and nutrient digestibility

Abstract

This study aimed to evaluate the effects of an organic acid blend and essential oils individually and in combination on growth performance, blood biochemical profile, gut health, and nutrient digestibility of broilers fed a higher level of an animal protein concentrate. Five hundred day-old Ross-308 male broiler chicks (average body weight, 39 ± 1.2 g) were randomly assigned to five replicated (5 replicates/treatment; 20 birds/replica) dietary treatments (100 birds/treatment). Birds in these group were given five different experimental diets that were prepared and designated as (i) basal diet (negative control, NC); (ii) basal diet plus Enramycin (positive control, PC), 50 mg/kg feed; (iii) basal diet with the addition of organic acid (OA) at 200 mg/kg feed; (iv) basal diet plus essential oils (EO) at 150 mg/kg feed; (v) basal diet plus combination of OA and EOs 200 and150 mg/kg feed (OA + EO). Experimental diets were prepared and fed in two phases i.e. starter (1–21 d) and finisher (22–42 d) phases of rearing. The findings of the present study revealed that feed intake did not vary significantly among the treatment groups, however, better (p < .05) body weight gain (BWG) and feed conversion ratio (FCR) was observed in OA, EO and OA + EO supplemented groups, respectively. The growth of Clostridium perfringens, Escherichia coli and Salmonella were reduced (p < .05) in OA, EO, OA + EO groups, while, Lactobacillus growth was positively improved (p < .05). Notably, the intestinal lesion score was significantly reduced, and villus height was improved in the OA, EO, OA + EO groups compared to birds in the NC group. Moreover, the serum level of calprotectin and liver enzymes were significantly reduced in the OA + EO treated group. At the end of the trial five birds from each experimental replicate of all five treated groups were shifted to metabolic cages on day-36 till day-42 for daily excreta collection and euthanized for ileal digesta to asses apparent metabolisable energy AME and nutrients digestibility. All dietary treated groups compered to birds in the NC group enhanced nutrients digestibility and AME. It was deduced that supplementing organic acids and essential oils are beneficial in improving birds performance, health, nutrient digestibility and subsequently could potentially replace antibiotic as growth promoters and could enhance the utilisation of animal protein concentrate without compromising performance and gut health in the poultry feed industry.

HIGHLIGHTS

• Supplementation of diet with organic acid and essential oils are potential alternative to antibiotic growth promoters

• Organic acid and essential oils in combination were more useful for growth performance, nutrient utilisation and gut health of broilers

Keywords:

• Broilers
• feed additives
• nutrients bioavailability
• gut health
• antibiotics

Introduction

Advancement in nutrition, genetics, managemental practices and housing has led to tremendous contributions to the poultry sector across the globe (Korver 2023). A number of nutritional interventions including the use of antibiotics have played a tremendous role in enhancing the body growth rate and gut health of broiler chickens (Abd El-Hack et al. 2020; Jin et al. 2020). There is always a growing concern on the indiscriminate use of antibiotics in poultry production due to antimicrobial resistance. This has led to a reduction and or complete ban on the use of antibiotics in poultry production to ensure quality products and to safeguard public health (Sultan et al. 2023). However, to sustain an optimum growth rate and gut health of poultry birds potential alternatives are needed to be investigated to maintain improved productivity (Robinson et al. 2019). Similarly, alternative and cheaper source of protein to replace soybean meal has also grabbed considerable attention to minimise poultry feed costs. However, the replacement of antibiotics and higher inclusion rate of less digestible alternative protein sources could lead to compromised performance, gut health issues and losses. Acidifiers that are commonly known, as organic acids are present naturally have been reported to significantly reduce the growth of harmful bacteria and fungi and are commonly used as food preservatives to minimise food contamination (Adewole et al. 2021). Organic acids can potentially suppress the growth pathogenic bacteria (Pham et al. 2022) and help to reduce the pH in the gastrointestinal tract, enhance pepsin activity and assess to improve nutrients digestibility (Dibner and Buttin 2002). It helps to avert the impacts of toxic compounds released by certain bacteria and its colonisation thus preventing intestinal epithelial cell damage and improve villus height (Adewole et al. 2021). It is well established that organic acids (OAs) addition in animal feed and or drinking water could provide chickens with protection against certain harmful microbes like E. coli, Salmonella, C. perfringens and Campylobacter infection (Ebeid and Al-Homidan 2021). The mode of action of OAs is by penetrating the bacterial membrane, inhibiting ATP synthesis, and or disrupting the bacterial membrane and denaturing the DNA (Mroz et al. 2006; Nguyen et al. 2020). Numerous studies have reported that OAs has the potential to selectively inhibit the growth of pathogenic microbes while preserving beneficial microbe populations, thus creating a eubiotic intestinal environment (Pryde et al. 2002; Carrasco et al. 2019; Jackman et al. 2020).

Essential oils (EOs), such as thymol, carvacrol, and eugenol, are plant derived mixture of potent compounds that exhibit growth and gut health promoting benefits in poultry (Bassolé and Juliani 2012; Stevanovic et al. 2018). Moreover, essential oils possess anti-inflammatory (Craig 2001), immunomodulatory (Szigeti et al. 1998) and antioxidative (Fernandez-Panchon et al. 2008) properties that helps birds to maintain good health in stressful conditions (Jang et al. 2007), which result in enhanced production performance and health of chickens (Kazempour and Jahanian 2017). Plant-derived essential oil such as thyme, carvacrol, cinnamaldehyde and citral have been demonstrated in vitro to either inhibit or kill Gram-negative and Gram-positive bacteria including Salmonella, E. coli, Campylobacter and Clostridium perfringens, without compromising the colonies of beneficial bacteria (Calo et al. 2015; Do et al. 2015; Lopez-Romero et al. 2015; Yang et al. 2015)

It is highly imperative to carefully monitor the inclusion rates and sources of protein in poultry feed to maintain a healthy and balanced gut microbiota and to promote optimal animal performance (Gilani et al. 2013). The use of animal protein source in poultry diets is highly effective in minimising cost but at the same time can compromise the production performance and gut health of the birds if not handled carefully (Zanu et al. 2020). There is potential scope to incorporate cheaper animal protein sources in poultry diet with OAs and essential oils since both have beneficial effects on gut health and performance synergistically (Choi et al. 2022). Presently in the local poultry feed sector of Pakistan animal protein concentrates are included in the poultry diets variably up to 3–4% with no or minimum adverse effects on poultry health and performance. There is however, a dearth of information on the higher inclusion rate (6%) of animal protein concentrate (APC), that is locally produced in Pakistan from poultry by-products and have poor and variable quality in terms of processing. Therefore, a need for deeper understanding was felt to assess the individual and combined effects of organic acids and essential oils with a higher inclusion rate of APC (6%) to fully exploit it beneficial implications.

Materials and methods

Dietary treatments and birds husbandry

Five iso-caloric and iso-nitrogenous experimental diets were prepared that included 6% of animal protein concentrate using Brill V. 2.0 ration formulation software as per standard requirements of Ross-308 nutritional guides (Table 1). Chromic oxide was added to all diets during the finisher phase to serve as an indigestible marker. The first diet was designated as negative control (NC) that had no additive in it, a second diet called positive control (PC) was supplemented with Enramycin (50 mg/kg; commonly used antibiotic as AGP in local poultry). The third and fourth diets were supplemented with a commercial organic acid blend OA (200 mg/kg) and a coated mixture of essential oils EO (150 mg/kg), respectively as per the standard recommendation of the producer. The fifth diet contained a combination of both OA and EO (200 and 150 mg/kg, respectively). The commercial organic acids carried formic acid (20.7%), propionic acid (12.8%), ammonium formate (17.5%), and ammonium propionate (4.2%) and amorphous silica (CAS No: 112926-00-8) as a carrier. The coated EOs supplement contained thymol and carvacrol (7.5 g kg⁻¹ each of the commercial product) and a carrier.

Table 1. Composition and estimated nutrient values of experimental diets.

Ingredient %	Starter (1–21 d)	Finisher (22–42 d)
Corn	59.820	61.910
46% Soybean meal	28.410	26.220
APC	6	6
Poultry Oil/Fat	1.840	2.630
NaCl	0.350	0.350
NaHCO3	0.100	0.100
Limestone	1.450	1.321
Di Calcium Phosphate	0.870	0.640
Lysine Sulfate	0.390	0.2510
DL Methionine	0.320	0.220
Threonine	0.110	0.010
70% Choline Chloride	0.100	0.100
Mineral and Vitamin Premix	0.250	0.240
Phytase	0.010	0.010
Calculated nutrients
Dry matter	85.283	83.711
Protein	21.051	19.430
Ash	5.199	4.669
Ether extract	5.121	6.702
Crude Fiber	2.714	2.641
AME (Kcal kg^−1)	2975	3100
Na	0.180	0.180
Cl	0.283	0.285
K	0.849	0.796
Calcium	0.900	0.760
Available Phosphorus	0.450	0.380
Lysine (D)	1.220	1.020
Methionine (D)	0.597	0.501
Met + Cys (D)	0.311	0.800
Tryptophan (D)	0.223	0.207

Values are presented in percentage, otherwise stated.

AME: Apparent metabolisable energy; APC: Animal protein concentrate.

A total of 500 Ross-308 male day-old broiler chicks were procured from a local commercial hatchery and were randomly allocated to different dietary treatments (n = 5) as above and each dietary treatment was further replicated (n = 5) carrying 20 birds per replicate (100 birds per treatment group). Birds were shifted to individual pens (5 L × 5 W × 2H feet) that served as replicate in an open sided house provided with an optimum environmental conditions and provision of quality wood shavings as bedding material, drinkers, feeders and heating source inside each pen. The temperature in each room was set at 33 °C on the day of bird placement, and subsequently temperatures were reduced by 0.75 °C for each of the first 7 D and then by 0.42 °C per day until 21 °C where it remained for the rest of the trial. The lighting program and light intensity were 23 h light 1 h dark (23 L:1D) at 20 lux for 0 to 2 D, 22 L:2D at 18 lux from 3 to 4 D, 21 L:3D at 16 lux from 5 to 6 D, 20 L:4D at 14 lux for 7 to 8 D, 19 L:5D at 12 lux from 9 to 10 D, 18 L:6D at 10 lux from 10 to 11 D, and 17 L:7D at 10 lux for the remainder of the trial. Humidity was maintained at 55–60%

Birds in all pens had an ad libitum access to feed and fresh drinker water. Feeding was practiced in two phases, a starter phase, crumble diet was offered from day-1 to 21 and a finisher phase pellet diet was given from day-22 to 42 days (Table 1). All birds in this study were vaccinated against ND (day-6 and 24), IB (day-6), and IBD (day-18).

Performance of birds

Body weight gain and feed consumption were recorded on a weekly basis to calculate average body weight gain, feed intake and feed conversion ratio (FCR) for each feeding phase. FCR was adjusted in each pen for mortality, if any.

Determination of ileal microbial count

Lower ileal digesta was collected from two random birds in each replicate pen and pooled at day- 21 and 35 for microbial counting of all five different treatments. Soon after collection, samples were immediately shifted to the clean-labeled tubes and placed at − 80 °C until analysis. Briefly, 1 g of the excreta sample was taken, and diluted in 1% peptone broth and homogenised to obtain a uniform suspension. Aliquots of the suspension were then added to selective media to promote the growth of specific bacterial species. Lactobacillus MRS Agar plates were used to isolate Lactobacillus species, MacConkey agar plates were used to isolate Escherichia coli and Salmonella-Shigella agar plates were used to isolate Salmonella species. The plates were incubated at the appropriate temperature and conditions for the specific bacterial species. To find out the growth of Clostridium perfringens the Clostridium perfringens selective agar was used. The media was incubated for 48 h. In each media, the respective growth of the bacteria was examined, and the colony was counted by colony counter (Gao et al. 2019; Vinolya et al. 2021).

Intestinal lesion scoring

Birds randomly selected (in two/replicate of all five different treatments); as above for ileal digesta collection (microbial count) were also used for lesion scoring. After the ileal digesta collection the entire intestine of each bird were taken out and opened aseptically to record the lesion score. The severity of the lesions was scored from 0 (no lesions) to 4 (severe lesions) based on the description and observations of Vinolya et al. (2021).

Blood haematology, liver health indicators and serum calprotectin analyses

On day 35 of the experimental period, blood samples from two birds per replicate were collected and analysed for various hematological parameters using an automatic blood-cell analyser (Adil et al. 2010). The remaining portion of the blood was centrifuged at 4 °C for 20 min at 4000 x g. The clear supernatant was collected from each group and stored at −40 °C for onward analysis of Alanine transaminase (ALT), and Alkaline phosphatase (ALP) using commercial kits (Chand et al. 2018), and for the determination of serum calprotectin (CALP) using commercial ELISA kit (Bortoluzzi et al. 2021).

Gut morphology

Ileum, duodenum, and jejunum segments (4 cm each) of two birds per replicate were taken and processed for histological analysis. The collected segments were then fixed in 10% neutral buffered formalin for 24 h, processed for paraffin embedding, and cut into cross sections of 4 μm thickness using a microtome (Accu-Cut SRM 200 Sakura). These sections were then stained with hematoxylin-eosin dye and examined under a light microscope. Four vertically oriented villi and their crypts of each group were examined, and the value of villus height and crypt depth was noted for each segment. Additionally, the height-to-crypt depth ratio was computed for each group (Choi et al. 2022).

Apparent ileal digestibility

At the end of the experimental trial five birds from each replicate were shifted to specially design metabolic cages fitted with aluminium trays for the collection of faecal material. Birds were shifted on day 36 till 42. During the last four days, daily feed consumption was measured and every next day faecal material was collected (four days) per replicate, weighed and kept in the freezer. On the final day of collection, faecal material from all replicates were pooled weighed and 20% of samples was take for further analyses. In addition, a known amount of Cr₂O₃ (0.5%) were added to the finisher diet to serve as an indigestible marker (Islam et al. 2022). On day-42 birds in all replicates were euthanized, the lower half of the ileum was identified, and its contents were flushed into plastic containers and pooled per replicate. The ileum was defined from the point of Meackle’s diverticulum to 2-3mm proximal to the ileo-ceacal junction. The sample of the feed, ileal digesta and excreta was oven-dried, ground to 1 mm particle size. The proximate analysis of the grounded feed and ileal digesta samples was done according to the AOAC (2005). Dry matter (DM) was measured as per protocol defined in the AOAC (1990) method (925.09) using an oven drying of 5.0 g sample at 105 °C. Nitrogen content of feed and ileal digesta was measured using the Kjheldhal method (990.03; AOAC, 1990) and CP was calculated as N 6.25. Ether extract in samples was determined after hexane extraction (Method 920.39; AOAC, 1990) in an Ankom extraction system (Macedon, NY). The gross energy of the feces and diet was determined by the adiabatic bomb calorimeter model (IKA, Werke C7000) (Stefanello et al. 2019). A spectrophotometer was used to determine the contents of Cr₂O₃ in feed, ileal digesta and faecal material (Williams et al. 1992). The nutrient digestibility coefficient and apparent metabolisable energy (AME) was determined as: $$\mathrm{\text{AME}}(\mathrm{\text{kcal}}/\mathrm{\text{kg}})=\left\{{\mathrm{\text{GE}}}_{\mathrm{i}}-\left(1+{\mathrm{\text{GE}}}_0\times \frac{{\mathrm{C}}_{\mathrm{i}}}{{\mathrm{C}}_0}\right)\right\}$$

GEi and GEo are the gross energy (kcal/kg) in the diet and digesta respectively; Co and Ci are the concentration of indigestible marker in diet and digesta respectively. $$\mathrm{\text{Digestibility}}\left(\mathrm{\%}\right)=\left\{\mathrm{ }1-\left(\frac{{\mathrm{C}}_{\mathrm{i}}}{{\mathrm{C}}_0}\times \frac{{\mathrm{N}}_0}{{\mathrm{N}}_{\mathrm{i}}}\right)\right\}\times 100$$

Co and Ci = represent the amount of indigestible marker in digests and diet respectively; Where Ni and No represent the nutrient content in the diet and digesta respectively.

Data analysis

The data obtained were stored and sorted in Excel, 2016 and analysed using a one-way analysis of variance (ANOVA) using General Linear Model in SAS (9.3 package) at a p-value of α = 0.05. Means of different parameters were compared using LSD (least significant difference) multiple comparisons test.

Results

Growth performance

Findings of the present study indicated that feed intake was not affected (p > .05) both in starter, finisher phases and overall, see Table 2. However, body weight gain and FCR was significantly improved (p < .05) differently in all treated groups both in the starter and finisher phases. Organic acids and essential oils both individually and in combination had a significant impact on improving the FCR compared to the antibiotic-treated group though insignificant among themselves. Similarly, the difference in body weight gain for essential oils and a combination of EO and OA treated groups was insignificant however significantly better to the rest of the treatments studied in the present trial.

Table 2. Effect of organic acids (OA) and essential oils (EOs) on the growth performance of broilers at starter and finisher phase.

Traits	Diets*
	NC	PC	OA	EO	OA + EO	SEM	p-value
Starter (1–21 d)
BWG, g	741.100^c	758.500^b	787.500^a	790.500^a	797.400^a	0.264	.000
FI, g	1123	1128	1126	1124	1124	0.041	.692
FCR	1.650^a	1.480^b	1.420^c	1.420^c	1.410^c	0.011	.049
Livability %	94.500	96.200	95.700	96.500	97.700	0.882	.673
Finisher (22–35 d)
BWG, g	1025^d	1043^c	1056^b	1069^a	1070^a	0.006	.006
FI, g	2240	2232	2238	2239	2239	0.321	.321
FCR	2.180^a	2.130^b	2.120^b	2.090^b	2.090^b	0.007	.007
Livability %	96.700	96.500	96.500	96.500	96.300	0.999	.999
Overall (1–35 d)
BWG, g	1766^c	1801^b	1843^b	1860^a	1867^a	0.023	.000
FI, g	3363	3360	3364	3363	3364	0.004	.358
FCR	1.900^a	1.860^b	1.820^b	1.810^b	1.800^b	0.034	.000
Livability %	94.500	97	97.200	96.500	97.300	0.037	.743

* NC: negative control group fed on standard basal diet; PC: positive control basal diet plus Enramycin at 50 mg/kg feed; OA basal diet with the addition of organic acid at 200 mg/kg feed; EO, basal diet plus essential oils at 150 mg/kg feed; OA + EO, basal diet plus combination of OA and EO at 200 and 150 mg/kg feed, respectively.

SEM: standard error of means; BWG: body weight gain; FI: feed intake; FCR, feed conversion ratio.

Means with different superscript represent significant difference at p < .05.

Ileal microbial count

Ileal microbial count for different treated groups is shown in Table 3. In the starter phase of the study, it appeared that the growth of E-coli, Salmonella, and Clostridium perfringens were reduced and increased Lactobacillus (p < .05) in groups that received antibiotics, OA, EO, and a combination of OA + EO to the group that did not receive any supplementation. A similar trend was seen in the finisher phase of this study for the microbial community. Lactobacillus count tended to improve significantly (p < .05) in OA, EO, and combination of OA + EO treated groups.

Table 3. Effect of organic acids (OA) and essential oils (EOs) on the ileal microbial count of broilers at day-21 and 35.

Gut microbes	Diets*
	NC	PC	OA	EO	OA + EO	SEM	p-value
Day-21
Clostridium perfringens	4.450^a	4.280^a	4.210^b	4.230^b	4.240^b	0.052	.005
Escherichia coli	7.490^a	7.290^b	7.270^b	7.260^b	7.190^c	0.105	.034
Salmonella	6.990^a	6.670^b	6.650^b	6.150^c	6.140^c	0.023	.000
Lactobacillus	8.350^c	8.700^b	8.880^a	8.890^a	8.990^a	0.014	.005
Day-35
Clostridium perfringens	4.940^a	4.130^b	3.970^bc	3.680^c	3.630^c	0.031	.002
Escherichia coli	7.710^a	7.410^b	7.400^b	7.280^b	7.170^c	0.051	.000
Salmonella	6.780^a	6.490^b	6.420^b	6.210^c	6.140^c	0.021	.000
Lactobacillus	8.450^c	8.570^b	9.880^a	9.900^a	9.960^a	0.102	.047

* NC, negative control group fed on standard basal diet; PC, positive control basal diet plus Enramycin at 50 mg/kg feed; OA basal diet with the addition of organic acid at 200 mg/kg feed; EO, basal diet plus essential oils at 150 mg/kg feed; OA + EO, basal diet plus combination of OA and EO at 200 and 150 mg/kg feed, respectively.

SEM: standard error of means; BWG: body weight gain; FI: feed intake; FCR: feed conversion ratio.

Means with different superscript represent significant difference at p < .05.

Intestinal lesion score observation

Broilers that were fed on a diet carrying 6% animal protein concentrates but were not treated with any feed additive exhibited severe intestinal lesions in the duodenum, jejunum, and ileum compared to the groups that either received antibiotic, OA, EO, and its combination of OA and EO supplementation Figure 1(A–C). Additionally, the study found that adding OA at 200 mg/kg and EO at 150 mg/kg significantly reduced the intestinal lesion score, indicating a positive impact on gut health.

Figure 1. (A–C). Effects of organic acids (OA) and essential oil (EOs) on the lesion score of duodenum, jejunum and ileum respectively at day 35.

Blood haematology, liver health indicators and serum calprotectin analyses

Observation made on hematological and serum parameters in different dietary treatments were given in Table 4. The hematological parameters including packed cell volume, red blood cell, and white blood cell, haemoglobin level, serum total protein, albumin, and globulin were not significantly affected by the treatments (p > .05). Liver function parameters ALT were increased both in NC and PC groups, an indicative of the adverse impact of animal protein concentrate and antibiotics that was significantly rectified by the addition of OA, EO and it combination. The content of CALP was significantly reduced by birds supplemented with both EO + OA (22.23 ng/dl), followed by EO treatment (24.34 ng/dl) with no difference between PC and OA treatments while the highest level was noticed by birds in NC group (28.50 ng/dl).

Table 4. Effect of individual or combination of organic acids and essential oils on blood haematology and serum biochemistry of broilers.

Parameters	Diets*						p-value
	NC	PC	OA	EO	OA + EO	SEM
PCV %	30.140	30.320	30.220	30.460	30.480	0.0760	.484
Hb mg/dl	11.620	11.750	11.430	11.520	11.700	0.134	.807
RBC (cells × 106/mm^3)	2.580	2.560	2.600	2.640	2.600	0.031	.891
WBC (cells × 103/mm^3)	10.370	10.380	10.410	10.470	10.440	0.041	.838
Total protein(g/dL)	3.380	3.530	3.370	3.410	3.450	0.031	.166
Serum Albumin (g/dL)	2.410	2.440	2.450	2.410	2.450	0.023	.061
Serum Globulin (g/dL)	2.280	2.250	2.260	2.320	2.290	0.004	.719
CALP (ng/dL)	28.580^a	26.500^b	26.670^b	24.340^c	22.230^d	0.821	.002
ALT (U/L)	261.140^a	247.790^b	239.170^c	235.630^c	234.520^c	0.727	.041
ALP (U/L)	9.030^a	8.760^a	7.430^b	7.520^b	7.430^b	0.069	.034

^A NC: negative control group fed on standard basal diet; PC: positive control basal diet plus Enramycin at 50 mg/kg feed; OA basal diet with the addition of organic acid at 200 mg/kg feed; EO, basal diet plus essential oils at 150 mg/kg feed; OA + EO, basal diet plus combination of OA and EO at 200 and 150 mg/kg feed, respectively.

ALT: Alanine transaminase, ALP: Alkaline phosphatase; SEM: standard error of means; Means with different superscript represent significant difference at p < .05.

Intestinal morphology and morphometric analysis

It was interesting to note that birds that received a diet with no added feed additives had poor villus height and crypt-depth ratio that was significantly improved when birds were supplemented with OA, EO and their combination in other groups except on day-21 and day-35 where the difference among NC, PC and OA group was insignificant for duodenum and jejunum, respectively. These beneficial impacts of feed additives were seen in both the starter and finisher phases (Table 5).

Table 5. Effect of individual or combination of organic acids and essential oils on gut morphology of broilers.

Gut section	Diets^a					SEM	p-value
	NC	PC	OA	EO	OA + EO
Duodenum	Day-21
VH (µm)	1067^b	1068^b	1076^b	1134^a	1144^a	0.124	.000
CD (µm)	108.300^a	107.300^a	98.160^b	94.660^c	97.330^b	0.234	.000
VCR	9.850^c	9.960^c	10.960^b	11.980^a	11.800^a	0.035	.000
Jejunum			Day-21
VH (µm)	1043^c	1045^c	1064^b	1061^b	1117^a	0.811	.000
CD (µm)	134.500^b	135.100^a	133.200^b	134.700^b	135.800^a	0.650	.000
VCR	7.750^c	7.740^c	7.990^b	7.880^b	8.220^a	0.032	.000
Ileum			Day-21
VH (µm)	552^d	552.100^d	590.500^c	675.700^b	723.500^a	0.540	.000
CD (µm)	104.700^a	101.800^a	97.000^b	95.200^b	89.500^c	0.432	.000
VCR	5.280^d	5.420^d	6.080^c	7.100^b	8.080^a	0.063	.000
Duodenum			Day-35
VH (µm)	1124^c	1189^b	1185^b	1246^a	1256^a	0.781	.000
CD (µm)	113^a	114.800^a	112.700^a	106^b	106.500^b	0.490	.000
VCR	9.950^c	10.400^c	10.500^b	11.800^a	11.800^a	0.271	.000
Jejunum			Day-35
VH (µm)	1191^c	1191^c	1190^c	1213^b	1305^a	0.231	.000
CD (µm)	172.200^a	172.500^a	152.200^c	153.700^b	145.700^d	0.481	.000
VCR	6.910^d	6.900^d	7.820^c	7.890^b	8.950^a	0.021	.000
Ileum			Day-35
VH (µm)	670^d	669.5^d	710.7^c	738^b	815^a	0.034	.000
CD (µm)	120.800^a	120.100^a	114.300^b	111.700^c	101.500^d	0.760	.000
VCR	5.540^d	5.570^d	6.210^c	6.610^b	8.030^a	0.031	.000

^a NC: negative control group fed on standard basal diet; PC: positive control basal diet plus Enramycin at 50 mg/kg feed; OA basal diet with the addition of organic acid at 200 mg/kg feed; EO, basal diet plus essential oils at 150 mg/kg feed; OA + EO, basal diet plus combination of OA and EO at 200 and 150 mg/kg feed, respectively.

VH: villus height; CD: crypt depth; VCR: villus height-to-crypt depth ratio; SEM: standard error of means; Means with different superscript represent significant difference at p < .05.

Ileal nutrient digestibility and apparent metabolisable energy

It was obvious that the ileal digestibility of different nutrients was compromised in birds fed on animal protein concentrates without any feed additives. However, this was significantly enhanced by birds in groups that were treated with OA, EO and it combination. Apparent metabolisable energy was also improved by all dietary treatments compared to birds in the negative group, presented in Table 6.

Table 6. Effect of individual or combination of organic acids and essential oils on nutrient digestibility and apparent metabolisable energy of broiler.

Digestibility %	Diets*						p-value
	NC	PC	OA	EO	OA + EO	SEM
Dry matter	76.400^c	76.300^c	77.900^b	78.980^a	79.900^a	0.043	.000
Crude protein	76.960^d	78.950^c	79.460^c	81.900^b	83.460^a	0.037	.000
Ether extract	88.300^c	90.500^b	90.300^b	91.300^a	91.300^a	0.240	.000
AME	2699.900^c	2735^b	2768^b	2847^a	2888^a	0.160	.000

* NC: negative control group fed on standard basal diet; PC: positive control basal diet plus Enramycin at 50 mg/kg feed; OA basal diet with the addition of organic acid at 200 mg/kg feed; EO, basal diet plus essential oils at 150 mg/kg feed; OA + EO, basal diet plus combination of OA and EO at 200 and 150 mg/kg feed, respectively.

AME: apparent metabolisable energy; SEM: standard error of means; Means with different superscript represent significant difference at p < .05.

Discussion

Findings of the present research study revealed that supplementation of essential oil EO, organic acid OA, their combination and antibiotic had a positive impact on growth performance, nutrient digestibility and gut health of birds that were fed a higher level (6%) of animal protein concentrate. Poultry by-product meal (locally known as animal protein concentrate in the poultry industry) is the resultant of processing poultry feathers, shanks, head, neck and intestine and other non-edible parts. This product is highly variable in quality depending on composition and processing conditions. The digestibility of protein of animal protein concentrate have been reported to be low up to 50–55% and the majority of it goes undigested that results in hind gut fermentation and other gut health related issues. Hind gut fermentation of the undigested protein could result to ammonia, polyamine, indoles, and skatole formation, that in turn could adversely impact gut health and animal performance (Qaisrani et al. 2015; Apajalahti and Vienola 2016). The presence of undigested protein fraction at the distal tract of poultry birds could enhance the level of certain specific amino acids that could lead to alternation in normal gut microbiota and susceptibility to various intestinal disease (Dahiya et al. 2007). However, it is pertinent to mention that the use of animal protein concentrate is still considered as an option to minimise poultry feed cost by replacing expensive soybean meal by the field poultry nutritionist locally and is used differently in different poultry diets. To curb the untoward consequences associated with feeding of high level of animal protein concentrate in poultry diets, a number of strategies are opted. This include the use of antibiotics to promote growth and maintain good gut health (Mehdi et al. 2018) that is now discouraged and is banned in many countries (Adewole et al. 2021), organic acid blends, coated essential oils and their combination. In this study improved body weight gain and FCR by birds in groups treated with organic acids, essential oils or their combination could be attributed to it potential keeping good gut health by reducing the number of harmful microflora (Dhawale 2005), maintaining pH and increased surface area for nutrients assimilation (Gharib-Naseri et al. 2012). Vinolya et al. (2021) observed similar findings with the addition of organic acids and essential oils fed to broiler birds and support the findings of this study. Novel coated essential oils used in the present study carrying carvacrol and thymol, are known to have strong antimicrobial effects against a wide range of bacteria, including E. coli and Salmonella (Scicutella et al. 2021). Antibiotic treated group performed nearly the same as with OA and EO or their combination confirming that its replacement with such additives could be achieved to minimise the antimicrobial resistance. Studies have shown that chickens are commonly exposed to bacterial pathogens, such as Escherichia coli and Salmonella spp., which can cause gastrointestinal disorders and adversely affect their nutrient digestion and absorption capacity (Crisol-Martínez et al. 2017). The EOs has been shown to possess antimicrobial properties, which may help to control the growth and colonisation of pathogenic bacteria in the gut of broiler chickens. This can reduce inflammation and competition with pathogenic microbes, which in turn can improve feed efficiency by promoting better nutrient digestion and absorption (Nava et al. 2009; Adil et al. 2011). Findings similar to present investigations were also reported by Pirgozliev et al. (2019) who reported that the addition of EOs carvacrol, cinnamaldehyde, and capsicum oleoresin to broiler chicken diets resulted in improved feed efficiency by reducing the growth of pathogenic bacteria and promoting the growth of beneficial gut bacteria.

The results of this study showed that dietary supplementation of OAs and EOs led to a reduction in the growth of pathogenic bacteria, including Salmonella, E. coli, and Clostridium, while promoting the growth of beneficial bacteria such as Lactobacillus. This suggests that these natural compounds could be used as an alternative to antibiotics in promoting a healthy gut microbiome in broiler chickens, that in turns improving the animals’ overall health and growth. Our findings are supported by the results of previous studies, who observed positive effects of OAs and EOs on the gut microbiome due to their ability to inhibit the growth and proliferation of undesirable opportunistic pathogens such as Salmonella, E. coli, and Clostridium, while promoting the growth of beneficial microorganisms like Lactobacillus (Jha et al. 2020; Pham et al. 2022; Ebeid and Al-Homidan 2021).

Improvement in intestinal health in term of villus height and villus: crypt depth ratio and reduction in the lesion scores suggested that supplementation with OAs and EOs may have a protective effect on the gut mucosa, reducing intestinal inflammation and improved intestinal integrity. This is consistent with previous studies that have reported improved gut health and decreased gross intestinal lesion scores in broiler chickens infected with necrotic enteritis and fed diets supplemented with essential oils or organic acids (Pham et al. 2022). Other similar findings were reported by (Abdelli et al. 2020; Stefanello et al. 2019).

Present findings suggested that the supplementation of EO, OA and its combination did not significantly alter hematological and serum parameters however, a positive impact on the CALP levels was observed in broiler chickens. A reduction in the liver enzymes ALT and AST was also noticed in treated groups that depicts that adverse impacts associated with feed high level of undigestible protein in the form of animal protein concentrate could influence liver function and this could be ameliorated by using organic acids and coated essential oils. Blood indicators are used to evaluate the health and nutritional status of broiler chickens (Torki et al. 2015). During inflammation, different types of blood cells such as heterophils and macrophages release various biomarkers, including cytokines, acute-phase proteins, and immunoglobulins. These biomarkers can be detected in the blood or faeces and are often used as indicators of inflammation and immune response in animals (Dal Pont et al. 2021). CALP, or calprotectin, has been shown to be a reliable biomarker of intestinal inflammation. The reduction in serum concentration of CALP in the groups fed with OAs, EOs, and the combination of OA + EO in the study suggests that these natural compounds may have anti-inflammatory effects in the intestine. These results are consistent with the findings of a previous study by Bortoluzzi et al. (2021), who also reported the CALP was reduced due to OAs and EOs supplementation. Reducing intestinal inflammation is essential for maintaining gut health and overall animal health. Therefore, the reduction in serum concentration of CALP by the supplementation of organic acids and essential oils blends may indicate a potential beneficial effect on the intestinal health of broilers (Dal Pont et al. 2021).

Improved villus height specifies a larger surface area for absorption of nutrients, while a shorter crypt depth suggests a more mature gut (Gong et al. 2021). Although the jejunum is the primary site for nutrient absorption, the ileum also plays an essential role in nutrients assimilation (Islam et al. 2022). According to our analysis, the OA, EO and EO + OA supplemented groups had higher VH: CD ratio than other groups. The beneficial effects of OAs and EOs on the intestinal morphology of birds are mainly due to their antimicrobial properties (Liu et al. 2017). A higher VH: CD ratio indicates that the villi are taller relative to the depth of the crypts, which suggests that there is more surface area available for nutrient absorption. Additionally, a shorter CD suggests that the gut is well developed, as the crypts become shallower with age (Ząbek et al. 2020).

The addition of feed additives, such as OAs and EOs, can improve the apparent ileal digestibility of various nutrients, including dry matter, crude protein, ether extract, and apparent metabolisable energy. This improvement is thought to be due to the ability of these additives to modulate gut microbiota, decrease pathogenic bacterial populations, and enhance the function of the digestive tract. Feed additives have been shown to increase the synthesis and activity of digestive enzymes, as well as the production of bile acids, leading to improved nutrient digestion and absorption in broiler chickens. In addition, EOs have been shown to increase the activity of trypsin and amylase, two important digestive enzymes involved in the breakdown of proteins and carbohydrates, respectively (Zaikina et al. 2022). Plant feed additives contain bioactive compounds, such as essential oils, phenolic compounds, and saponins, that can stimulate the secretion of digestive enzymes in animals. For instance, essential oils such as thyme, oregano, and rosemary have been shown to increase the activity of pancreatic enzymes in broiler chickens, leading to improved nutrient digestion and absorption

Conclusions

The study provides valuable insights into the potential benefits of feed additive comprising of organic acids and essential oils as alternatives to antibiotics in broiler chicken diets and the potential use of animal protein concentrates in poultry diet without compromising performance and gut health. The results demonstrated that the addition of organic acids and essential oils in broiler chicken diets could improve growth performance, gut health, and nutrient digestibility. The reduced pathogenic bacteria load and increased colonisation of beneficial bacteria in the digestive tract were highly valuable that might have contributed to improved animal health and improved performance. These results confirmed that organic acids and essential oils could be used as an alternative to antibiotics in the poultry feed carrying higher level of animal protein source. However, it warrants further investigations to examine the efficacy of such additives under different challenging conditions of stress with or without higher level of less digestible protein sources.

Ethical approval

The research study was prior approved for the procedures involving bird’s welfare and handling by the Ethical and Care Committee of the University of Agriculture Peshawar, Pakistan (No. PS-372_UAP_Pak).

Acknowledgments

The authors greatly acknowledge and express their gratitude to the Researchers Supporting Project number (RSP2024R462), King Saud University, Riyadh, Saudi Arabia.

Disclosure statement

The authors declared there is no conflict of interest

Data availability statement

The data analysed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

This research was supported by King Saud University, Riyadh, Saudi Arabia, Project number (RSP2024R462).