Effect of Organic Acids Blend, Micro- Encapsulated Phyto-Essential Oils Individually or in Combination on Growth Performance, Gut Health and Nutrients Utilization of Broilers

1Department of Poultry Science, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan 2College of Veterinary Sciences, Faculty of Animal Husbandry and Veterinary Sciences, The University of Agriculture Peshawar, Pakistan Article Information Received 14 July 2021 Revised 21 August 2021 Accepted 15 September 2021 Available online 27 December 2021 (early access)


INTRODUCTION
B roiler production performance is highly related to the gastrointestinal tract due to its vital role in the nutrient digestion and utilization (Rinttila and Apajalaht, 2013) and any deviation from normal gut function can badly impact the growth performance of broiler birds (Morgan, 2017). Antibiotic growth promoters (AGPs) have been extensively used in broiler production from improved growth performance and gut health . This however, could lead to antimicrobial resistance (AMR) (Golkar et al., 2014;Pourmand et al., 2017) and sever human health consequences in long run (WHO, 2014). It has been reported that if the use of AGPs

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beneficial implications e.g., antibacterial, antiviral, antifungal, antioxidant, anti-inflammatory, and immuneregulating properties (Swamy et al., 2016) could be effectively used to enhance the growth of poultry production. Certain essential oils thymol, carvacrol and eugenol can potentiate the growth performance and improve gut health (Henri and Bassole, 2012) and can modulate positively the microbial community of intestinal tract by favoring beneficial microbial growth and reducing harmful microorganisms (Stevanovic et al., 2018). These essential oils can stimulate gut digestive enzymes to improve nutrients digestibility, reduce the inflammatory response and enhance immunity (Kim et al., 2013;Kazempour and Jahanian, 2017). Organic acid are weak and short chain acids viz acetic acids, propionic, benzoic and butyric acids (Dibner and Buttin, 2002) that can effectively reduce gut pH favoring the growth of beneficial gut microbes, improve digestive enzymes functions and nutrients utilization by broilers (Canibe et al., 2001). Outside the digestive system supplementation of organic acids either in drinking water or feed have been reported to decrease pH of water and feed, reduce the growth harmful microbes and maintain its quality (Jarquin et al., 2007;Islam, 2012). Improved gut health of poultry birds with minimum number of harmful bacteria and better lining for the assimilation of nutrients could be achieved with the use of organic acids (Paul et al., 2007). There is great need to examine the potential benefits of organic acids blend and phyto essential oils in combination for effective replacement of antibiotic growth promoters in poultry diet. The combined effect of organic acids blend and phyto essential oils could have synergistic effect (Omonijo et al., 2018) to enhance the growth performance, immunity and nutrients utilization. It is therefore this study was designed to investigate the individual and combined impact of an organic acids blend and essential oil against the Zinc Bacitracin (ZB) that is commonly used as antibiotic growth promoter in poultry feed industry.

Ethical consideration
All the experimental procedures adopted in this study were pre-approved from the animal welfare and care committee on the use of experimental animals at The University of Agriculture, Peshawar, Pakistan.

Feed additives
Organic acid blend (propionic acid, formic acid, 2 Hydroxy 4 methyl thiobutanoic acid HMTBa) and microencapsulated phyto-essential oils blend containing (oregano, rosemary, cinnamon, and chili pepper extract) as active ingredients (both USA origin) were procured from a commercial feed additive supplier. Feed additives were mixed with micro-ingredients before mixing in final ration for better and uniform mixing.

Experimental layout and bird's husbandry
A total of 600 (day-old) broiler chicks (Cobb 500) were obtained from a commercial hatchery and reared in open sided house bedded with softwood shavings. All chicks were randomly assigned to five replicated (n=6; 20 birds/rep) dietary groups as CON; without any additives; ZB-150; OA-200; EO-150 and OA + EO, respectively. Birds in CON group was offered a starter (0-21days) and finisher (22-35 days) corn-soybean meal based diet fulfilling all its nutritional requirements as per Cobb 500 nutrients specification guide using Brill Formulation ® software (Table I), while the birds in others groups were added Zinc Bacitracin (150 mg/kg diet), organic acids (200 mg/kg feed) micro-encapsulated phyto-essential oil (150 mg/kg feed) and combination of organic acid (OA) and micro-encapsulated phyto-essential oil (EO) (200 mg + 150 mg/kg), respectively given. Birds in all groups had ad libitum access to feed and water. Optimum environmental conditions of temperature, humidity, ventilation and light were maintained as needed at different stages of rearing.

Data collection and measurements
Birds were weighed on day first and then on weekly basis. Initial weight was subtracted from final weight and divided by number of birds for that particular week and adjustment in mortality if any. Cumulative average body weight gain was measured by sum up all weekly weight gains. Feed intake per replicate was determined on weekly basis and cumulative was determined by summing up all data at the end of each phase. From the cumulative average body weight gain and feed intake feed conversion ratio (FCR) was measured (Sultan et al., 2018).

Relative weight of the lymphoid organs
On day 35, five birds from each replicate were randomly selected and live body weight was recorded. All birds were humanly killed, skinned off and dissected. Lymphoid organs thymus, spleen and bursa of Fabricius was carefully removed, trimmed of any foreign tissues and weighed individually and expressed as percent of the live body weight (Yang et al., 2018).

Determination of gut pH in different segment of the gastrointestinal tract
The gut pH was measured at four different segments of gastrointestinal tract i.e., crop, ilium, jejunum and ceca immediately after killing the birds. A portable digital pH meter was used as described by (Ndelekwute et al., 2019).

Determination of ileal microbial count
Ileal digesta content were collected both at day 21 and 35 of the experimental period from five birds and pooled. Samples were transferred to sterile plastic air tight test tubes, freezed and stored at -80°C until further analysis. Briefly, for measurement of ileal microbiota 1-gram excreta was diluted in 9 mL of 1% peptone broth and homogenized. Homogenized samples were transferred to selective media for growth. The bacterial counts was performed by serial 10-fold dilutions (10 g/l peptone solution) onto Lactobacillus MRS agar plates, MacConkey agar plates, and Salmonella-Shigella agar plates to isolate the Lactobacillus, Escherichia coli, and Salmonella, respectively. The bacteria colonies was counted immediately after the plates was cultivated at 37°C under anaerobic conditions using a colony counter (Gao et al., 2019).

Apparent ileal digestibility and ileal digestible energy calculation
Birds were selected (n=10) from all replicates at day-35 and shifted to metabolic cages for total excreta collection for final four days till day-42. Feed intake and excreta were collected and weighed daily morning for four days. Representative samples were collected, air and oven dried for further analyses. For determination of ileal nutrients digestibility 0.2% Cr 2 O 3 was used as indigestible marker. Dried samples of excreta, ileal digesta and feed was grind to pass through a 1-mm screen. Gross energy of feed and fecal samples was measured using Adiabatic bomb calorimeter and AME was determined. Proximate analyses of feed and excreta samples were done as outlined in (AOAC, 2005). Chromium concentrations were determined with a UV absorption spectrophotometer (Shimadzu, UV-1201, Shimadzu, Kyoto, Japan) using the method of (Williams et al.,1962). The following formulas were used to calculate the apparent ileal digestibility and ileal digestible energy (Stefanello et al., 2020).
where GEi= gross energy (kcal/kg) in the diet; GEo= gross energy (kcal/kg) in the ileal digesta or excreta; Ci and Co = concentration of marker in the diet and digesta or excreta (%), respectively.
where ND= nutrient digestibility (%); Ci and Co= concentration of marker in the diet and digesta or excreta (%), respectively; Ni and No= concentration of nutrient in the diet and digesta or excreta (%), respectively.

Data analysis
Data were subjected to one-way ANOVA using Effect of Organic Acids Blend, Micro-Encapsulated Phyto-Essential Oils Individually

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General Linear Model procedure of SAS 9.3 package (Guide, 2010). Treatments means were compared by LSD. Table II indicate improved average body weight gain and better FCR both at starter (day-1 to day-21) and finisher phase (day-22 to day-35) that was significantly (P <0.05) affected by all dietary treatments compared to control groups. During starter phase maximum average body weight gain was observed in groups OA+EO (796.91), , ) and improved FCR OA+EO (1.41), EO-150 (1.43), . This was followed by group ZB-150 and CON. Overall performance (day-1 to 35) in term of average body weight gain and FCR was improved (p<0.05) in all groups compared to control group. The highest final average body weight gain and better FCR were observed in the treatments groups ZB-150, OA-200, EO-150 and OA+EO as compared to CON. No significant difference (p>0.05) was seen among different groups for feed intake and livability.

Relative weight of lymphoid organs
Lymphoid organs weight, spleen and bursa of Fabricius were significantly improved (P < 0.05) in-group ZB-150, OA-200, EO-150 and OA+EO respectively. Maximum improvement in weight of spleen and bursa of Fabricius was observed in groups OA+EO as compare to other treatment groups. There was a non-significant difference (P > 0.05) between the treatments for the relative thymus weight Table III.

Digesta pH in different section of the digestive tract
pH in different gut section was significantly altered by the dietary treatments in groups that received organic acid blend, micro-encapsulated phyto-essential oil and or both. Maximum change was in pH in crop (4.94), proventriculus (2.36), ileum (6.01), Jejunum (5.12) and Caeca (5.91) was observed in group OA+EO as compared to control, Table  IV.

Ileal microbial count (log cfu g -1 )
Birds under different treatments showed a significant difference in microbial count of Escherichia coli, Salmonella and Lactobacillus both at starter and finisher phase of rearing, (Table V). Escherichia coli and Salmonella was found lowest in birds of group 6.85,6.10,6.70,respectively) and OA+EO (5.25,6.47) compared to birds in ZB-150 and CON group. However, the birds in same treatment groups had improved count of Lactobacillus (8.71,8.91,9.78) respectively, compared to other groups ZB-150 (6.16) and CON (7.68).

Nutrient utilization and ileal apparent metabolizable energy (Kcal kg -1 )
Table VI depicts findings of the nutrients digestibility and apparent metabolizable energy of all different treatments. It was interesting to note that digestibility of all different nutrients and energy utilization was significantly altered by organic acid, essential oils and or their combination with significant difference among these groups. Protein digestibility was maximum in group OA+EO (84.20%) and EO-150 (82.70%) compared to all groups with no significant difference among groups (ZB-150, OA-200 and CON). Significantly higher apparent metabolizable energy was recorded for groups OA+EO (2853.34 Kcal kg -1 ) and EO-150 (2821.71 Kcal kg -1 ), followed by group OA-200 (2756.23 Kcal kg -1 ) respectively. The antibiotic treated groups ZB-150 had no significant (p>0.05) impact however numerically higher values compared to controlled group.

DISCUSSION
The unrestricted use at sub-therapeutic levels of feed antibiotics as growth promoters may be associated with the development of antibiotic-resistant human pathogens (AGP) and therefore there is tremendous pressure on the poultry feed sector to phase out its use. Poultry experts across the globe are faced with the challenge of finding effective alternatives to replace AGP's for optimum poultry production. The use of essential oils and organic acids have shown substantial benefits in poultry production over the last few years (Banday et al., 2015). The additive effects of organic acids and essential oils have been observed on the gut health and growth performance in some previous studies (Liu et al., 2017). The variation of the gut microbiota may be the main mode of action linked to the synergic effects of a blend of organic acids and essential oils (Walia et al., 2017). The supplementation of organic acid and essential oil alone or in combination improved body weight gain in starter phase and overall production period in present study. The blend of organic acids and O n l i n e

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Z. Islam et al.  essential oils improve the body weight gain and feed efficiency at finisher phase of broiler production (Gheisar et al., 2015). A commercial blend of thyme, carvacrol and organic acids improve the feed conversion ratio and body weight gain in broiler chickens (Pham et al., 2020) and is related to present findings. The essential oils increase the permeability of bacterial membrane, which may expedite the influx of organic acid into the cytoplasm (Basmacioglu et al., 2016). It has been observed that dissociated forms of organic acid have the ability to reduce the intestinal pH and disturb the bacterial metabolism (Edgar and Oviado, 2019) that confirms outcomes of present study of reduced pH in organic acid supplemented group. Improved nutrients digestibly and energy utilization in present study could be attributed to a reduction in bacterial metabolism and population of pathogenic bacteria that could potentially promote digestion and nutrient utilization as has been reported previously (Ricke, 2003). A further improvement in the FCR in present study are similar to the finding of an earlier study in which a commercial blend of thyme, carvacrol and organic acids improved feed conversion ratio and body weight gain in broiler chickens (Yang et al., 2019). In the current study the blended organic acid and essential oil led to increase the development of spleen and bursa of Fabricius that indicate a better impact of these additives on lymphoid organs as has been observed previously (Sultan et al., 2015). Reduction in the digesta pH was significantly reduced by the diet containing organic acid and essential oil alone or their combination confer a better gut health and functioning of digestive tract in all different aspects. Ndelekwute et al. (2019) reported similar findings that supplementation of organic acid and essential oil reduce the gut pH in different sections of the gastrointestinal tract of chicken. Organic acid and essential has the ability to reduce the buffering capacity, thus lower the pH of the feed, and facilitate digestion in the intestinal tract (Garcia et al., 2008). Gut microbiota plays an important role for animal health, perfor mance, and product safety. Decreased

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Effect of Organic Acids Blend, Micro-Encapsulated Phyto-Essential Oils Individually 7 numbers of pathogenic bacteria in the gut may improve the ability of epithelial cells to regenerate villus and thus enhance intestinal absorption capacity (Zeng et al., 2015).
The current results showed that Escherichia coli and Salmonella population were decreased while Lactobacillus at 21-and 35 day-old broilers tended to be increased by encapsulated blends of essential oil and organic acid. The present results are in agreement with the findings of previous researchers (Gao et al., 2019). The essential oil and organic acid have antimicrobial potential which can be used against different pathogenic microorganism (Adewole et al., 2021). Organic acids and their salts decrease the digesta pH and constrains the replication of gram-negative bacteria like Escherichia coli, Salmonella (Rodjan et al., 2018). The decrease in population of pathogenic bacteria by organic acid and essential oil in chickens is associated with the changes produced by a blend of organic acids and essential oils, which may increase the bacterial resistance capacity of the intestine (Stanley et al., 2012). The use of organic acids and essential oils stimulate the pancreatic secretion and increase the gastric retention time, thus improve the nutrient digestion and absorption (Sethiya, 2016;Stamilla et al., 2020). Improvement in production performance, lymphoid organ weight, nutrients utilization and gut health implicate that organic acid and essential oils are more effective in poultry production.

CONCLUSION
The strategic supplementation of organic acids and essential oils either individually or in combination could significantly improve production performance of broiler birds. Moreover, a reduction in gut pH, harmful microbes and improved beneficial microbes indicates its impact as potential positive gut modulator that enable birds to utilize more nutrients from a given feed.

Statement of conflict of interest
The authors have declared no conflict of interest.

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Effect of Organic Acids Blend, Micro-Encapsulated Phyto-Essential Oils Individually