Antibiotic growth promoters (AGP) have been beneficial for bird’s health and productivity, but it is a double-edged sword (Miles et al., 2006). The rapid spread of drug-resistant pathogens and the public concerns over possible antibiotic residual problems as well as global concern over emergence of antibiotic-related environmental pollutants led to their ban by the European Commission (Haq et al., 2017). In view of this the researchers have to explore the utility of other non-therapeutic alternatives like organic acids, probiotics, prebiotics, herbs and essential oils as feed additives in poultry production (Khan et al., 2016). The organic acids penetrate the cell wall of pathogenic microbes, disrupt the normal cell functioning thereby causing death of microbes (Afsharmanesh and Pourreza, 2005; Mroz, 2005). Beside this, the organic acids do not form residue in meat or environment, preventing microbial resistance. In recent years, there has been increasing attention on the blending type (mixtures) of organic acids based on the assumption that synergistically positive effects of individual organic acid may exist (Kil et al., 2011). Hence, the experiment was designed to assess effect of organic acids and their combination on performance, gut health, immune status and economics as alternative to AGP.

Materials and Methods

Experimental Site

The experiment was carried out at Department of Poultry Science, COVAS, Parbhani, affiliated to MAFSU, Nagpur, India. The trial was conducted from of 18^th April to 29^th May 2018 which had the heat stress index ranging from 100.56^oF to 105.51^oF.   

Formulation of Experimental Ration

The quantity of feed ingredient required in the present experiment were purchased from local market.  Rations were prepared as per BIS (2007) at Feed Mixing Plant Department of Poultry Science, COVAS, MAFSU, Parbhani (Table 1). The pre-starter ration was offered for first seven days of age, starter ration was offered from 8^th day to 20^thday and finisher ration was offered thereafter up to 42^nd day of age. The iso-caloric and iso-nitrogenous feeding practices were followed. Organic acids (sodium citrate, ammonium formate and calcium propionate) and AGP (Bacitracin methylene disalicylate) were purchased from local market.

Experimental Birds and Data Collection                               

The experiment was approved by Institutional Animal Ethics Committee vide resolution no. IAEC/29/18 dated 11/05/2018. The experiment was carried out on 360 Vencobb⁴⁰⁰ day old straight run chicks for a period of 42 days. The chicks were weighed and distributed randomly into six treatment groups viz. A, B, C, D, E and F with four replicates of 15 chicks each. Experimental design used for housing of broilers is presented in Table 2. The standard feeding, watering, floor space and vaccination schedule were followed.

Table 1: Percent ingredient and nutrient composition of (basal diet) pre-starter, starter and finisher  rations with supplementation of organic acids

*Added in the diet as per the dose rate mentioned in experimental design

Table 2: Experimental design for housing of broiler with organic acids

*Bacitracin Methylene Disalicylate

Performance Parameters

The weekly live body weight, feed consumption, FCR, mortality and economics of broiler production were recorded replicate-wise.

Gut Health

pH of Different Parts of the Gastrointestinal Tract (GIT)                                                               

One bird per replicate was sacrificed on the 42^nd day of experiment. 1 g of gut content from crop, proventriculus, gizzard, duodenum jejunum and ileum were collected aseptically in 9 ml sterilized physiological saline (1: 10 dilution) and pH was measured using digital pH meter.

Immune Status

Antibody Titre against RD (Ranikhet Disease)

Serum sample from one bird per replicate was collected on the 7^th and 14^th day post R.D. vaccination. Antibody titre against RD (Ranikhet disease) was estimated in the laboratory of Poultry Diagnostic and Research Centre of Venkateshwara Hatcheries Pvt. Ltd., Pune.

Antibody Titre against Infectious Bursal Disease (IBD)

Serum sample from one bird per replicate was collected on the 7^th and 14^th day post I.B.D. vaccination. An indirect ELISA for IBD antibody test kit developed by PDRC (Poultry Diagnostic and Research Centre, Loni Kalbhor, Pune) was used. ELISA antibody titre against IBD (Infecitious Bursal Disease) was estimated at the laboratory of Poultry Diagnostic and Research Centre, Pune.

Statistical Analysis

Data, thus collected were subjected to statistical analysis by using Randomized Block Design by Snedecor, and Cochran, (2002).  The treatment means were compared by critical differences (CD) and analysis of variance.

Results and Discussion

Weekly Body Weight

The results from present study revealed significant (P <0.01) influence on weight for different treatment groups. The organic acids (ammonium formate, sodium citrate, calcium propionate and their combinations) were found to be superior to Bacitracin methylene disalicylate (BMD) in enhancing the live weight of experimental birds. It may be related to the reduction in pH in the gastrointestinal tract (GIT) and selective promotion of beneficial bacteria in the gut. The reduced pH is condensive for the growth of favorable bacteria, simultaneously hampering the growth of pathogenic bacteria which grow at a relatively higher pH. The organic acids may affect the integrity of microbial cell membrane or cell macromolecule or interfere with nutrient transport and energy metabolism in the pathogenic bacteria causing the bactericidal effect. The inhibition of intestinal pathogenic bacteria leads to the reduced metabolic needs, thereby increasing the availability of nutrients to the host. This also could decrease the level of toxic bacterial metabolites, causing an improvement in the protein and energy digestibility, thus improving the live weight of the birds (Adil et al., 2011). It indicates the growth promoting activity of organic acids and their combination. They may act as alternative to antibiotic growth promoter. These  results were supported by the findings of Samanta et al. (2009), Ghazalah et al. (2011), Hedayati et al. (2013), Sohail et al. (2015), Sultan et al. (2015), Tanzin et al. (2015), Bagal et al. (2016), Lakshmi et al. (2016), Ragaa et al. (2016), Rout et al. (2016), Kanagaraju et al. (2017) and Ramigani et al. (2017). In contrast to present study, Paul et al. (2007), Aghazadeh and Tahayazdi (2012) and Naveenkumar et al. (2017) reported non-significant influence on body weight by organic acids.

Feed Consumption

Table 3 and 4 revealed significant (P<0.01) influence on feed consumption. These findings are in close agreement with the results of Haque et al. (2010), Hudha et al. (2010) and Tanzin et al. (2015).

Table 3: Live bodyweight (g), feed consumption (g), FCR and mortality at different age groups                   supplemented with organic acids

Means bearing different superscript differ significantly row-wise

It may be attributed to the antimicrobial ability of organic acids. It enhances the digestibility of nutrients by improving the structure of intestinal flora (Tazoe et al., 2008). In contrast to present findings, Adil et al. (2011) and Ramigani et al. (2017) reported decreased feed intake and justified it with decreased palatability of diet.

Feed Conversion Ratio          

The FCR revealed significant (P< 0.01) differences (Table 3 and 4) in different treatment groups. An improvement in FCR may be due to increased feed intake by improving palatability of feed, increased permeability of mucosal cells of intestine followed by increased rate of utilization of nutrients resulting in increased body weight gain. Similar findings were observed by Chowdhury et al. (2009), Panda (2009), Adil et al. (2010),  Sohail et al. (2015), Bagal et al. (2016), Lakshmi et al. (2016) and Ramigani et al. (2017). In contrast to present findings, Rout et al. (2016) and Naveenkumar et al. (2017) recorded non-significant effect.

Mortality                                                                                                                                                  

There were no significant differences for mortality among different organic supplemented groups. These findings were in accordance with Hedayati et al. (2013), Lakshmi et al. (2013), Sultan et al. (2015), Kanagaraju et al. (2017) and Naveenkumar et al. (2017). They concluded that organic acid supplemented groups had mortality within normal range. It might be due to suppression of pathogenic bacteria by organic acids which led to healthy condition of birds throughout the experiment period. Similarly, Al-Natour et al. (2005) recorded a significant reduction in mortality in formic acid treatment group. Acikgoz et al. (2011) recorded numerically low mortality in organic acid treatment groups.

Table 4: ANOVA for body weight (g), feed consumption (g), FCR and at different age groups supplemented with organic acids

** Significant at 1% level.

Gut Health

pH of Different Parts of Gastrointestinal Tract

Significantly (P<0.01) decreased pH was recorded for crop (Table 5 & 6). It concludes that organic acids inhibit pathogen bacteria growth in the feed, at the opening of digestive tract thereby improving the performance of birds. Similarly, the pH of ileum differed significantly (P<0.05) for organic acid groups. The pH of provenrticulus, gizzard, duodenum and jejunum was numerically decreased but non-significant. The pH level in specific areas of the GIT is a factor which established a specific microbial population and also affected the digestibility and absorption of most nutrients. Most pathogens grow at a pH close to 7. The beneficial microorganisms live in an acidic media (5.8-6.2) in which they compete with pathogens. Hence, organic acids can be used as an alternative to antibiotic, increasing birds’ performance (Ghazalah et al. (2011).

Table 5: pH of different parts of gastrointestinal tract of broilers supplemented with organic acids

Means bearing different superscript differ significantly row-wise

Apart from antimicrobial and pH reducing effect, organic acids may have beneficial effect on the intestinal mucosa of broilers. As nutrient absorption in gut occurs from the intestinal mucosa, it may improve the nutrient utilization and consequently growth performance (Pelicano et al. 2005). These findings are supported by Ghazalah et al. (2011) and Ragaa et al. (2016), who observed that pH reduction was significantly greater in the upper part of GIT as compared to the lower part of the GIT. In contrast to the present findings, Salgado-Transito et al. (2011) and Marin-Flamand et al. (2014) reported non-significant differences in pH of GIT.

Table 6: ANOVA for pH values in different parts of the gastrointestinal tract of broiler supplemented with organic acids

**Significant at 1% level, * Significant at 5% level

Immune Status                                   

RD Titre

The differences between the overall antibody titre against Ranikhet disease (RD) at 14^th and 21^st day of age were non-significant (Table 7), however, it was numerically higher in sodium citrate and ammonium formate group. The findings in the present experimentation were in close agreement with the findings of Das et al. (2011), Marin-Flamand et al. (2014) and Deepa et al. (2017) who recorded non-significant differences in antibody titre against RD.  In contrast, Lakshmi et al. (2013), Sohail et al. (2015) and Fascina et al. (2017) recorded significant increase in antibody titre against RD for organic acid supplemented groups.

Table 7: Antibody titre against Ranikhet disease at 14^th day and 21^st day and Infectious Bursal disease at 21^stand 28^th day of age of broiler supplemented with organic acids

Means bearing different superscript differ significantly column-wise

IBD Titre 

Significant (P<0.01) influence on antibody titre of IBD at 21^st day was observed for calcium propionate (Table 7 & 8). These findings were in accordance with Ghasemi et al. (2014), Manafi et al. (2016) and Sohail et al. (2015) who found significant effect on antibody titer IBD. It indicates that broilers were healthy with higher immune status to fight against infectious diseases and enteric pathogens (Chowdhury et al., 2009). Similarly, calcium propionate and the combination of organic acids reported improved immune status of broilers. It indicates organic acids are capable of modifying gut microflora communities and therefore, might support improvement in immunity (Emami et al., 2013). However, differences at 28^th day for IBD titre were non-significant. It was in accordance with Lohakare et al. (2005).

Table 8: ANOVA of antibody titre against IBD at 21^st day of age for dietary organic acids

**Significant at 1% level

Economics of Broiler Production

Increased profitability per kg live weight (Table 9) was observed which is in agreement with findings of Haque et al. (2010), Tanzin et al. (2015) and Kamel and Mohamed (2016). It indicates more bioavailability of nutrients resulting in enhanced growth rate, superior FCR and ultimately more returns in the form of profit. Hence, organic acids could replace AGP (BMD) with no residual effect and optimum profitability of broiler production. In contrast, Chowdhury et al. (2009) and Rout et al. (2016) inferred a non-significant difference in profit between various treatments.

Table 9: Economics of broiler production supplemented with different organic acids

Conclusion

It is concluded that ammonium formate, sodium citrate, calcium propionate and their combination through feed were found to be beneficial for growth performance, gut health, immune status and economics. Hence, organic acids may be efficiently used as alternative to AGP (BMD).

Acknowledgement

The authors are highly grateful to Dr. N. M. Markandeya, Associate Dean, COVAS, MAFSU, Parbhani for providing necessary facilities and for his help at various stages of the experiment. The authors are also thankful to PDRC, Pune for providing serum estimation facilities.

References

1. Acikgoz , H.Bayraktar and O. Altan (2011)  Effects of formic acid administration in the drinking water on performance, intestinal microflora and carcass contamination in male broilers under high ambient temperature. Asian-Aus J. Animal Sci., 24(1):96–102.
2. Adil R., Q.M. Imtiyaz and T. M. Khan (2010) Effect of feeding organic acids on intestinal health of broilers. Poultry Sci., 31:54-58.
3. Adil S., M.T. Banday, G.A. Bhat, S.D. Qureshi and S.A. Wani (2011a). Effect of supplemental organic acids on growth performance and gut microbial population of broiler chicken. Livestock Res. Rural Dev., 23(1).
4. Adil S., M.T. Banday, G.A. Bhat, M. Salahuddin, M.RaquIb and S. Shanaz  (2011b). Response of broiler chicken to dietary supplementation of organic acids. Central Eur. Agric., 12(3):498–508.
5. Afsharmanesh, M., and J.Porreza (2005). Effects of calcium, citric acid, ascorbic acid, vitamin D on the efficacy of microbial phytase in broiler starters fed wheat-based diets: performance, bone mineralization and ileal digestibility. J. Poultry Sci., 4:418–424.
6. Aghazadeh A.M, M. Taha Yazdi (2012). Effect of butyric acid supplementation and whole wheat inclusion on the performance and carcass traits of broilers. Afr. J. Anim. Sci., 42:241-248.
7. Al-Natour and K. M. Alshawabkeh (2005). Using Varying Levels of Formic Acid to Limit Growth of Salmonella gallinarum in Contaminated Broiler Feed. Asian-Aust. J. Anim. Sci., 18 (3):390-395.
8. O.A.C. (2005). Official Methods of Analysis, 19^th Edn., Association of Official Analytical Chemists. Washington, USA.
9. Bagal L., V.K. Khatta,  B.S. Tewatial,  S.K. Sangwan and  S.S. Raut (2016). Relative efficacy of organic acids and antibiotics as growth promoters in broiler chicken. Veterinary World., 9: 377-382.
10. BIS (2007) Indian standard of poultry feeds specification. Bureau of Indian Standard, New Delhi, pp 14.
11. Chowdhury R., M.S. Islam, M.J. Khan, M.R. Karim, M.N. Haque, M. Khatun and G.M. Pesti (2009) Effect of citric acid, avilamycin, and their combination on the performance, tibia ash, and immune status of broilers. Poult. Sci., 88:1616-1622.
12. Das S.K., Islam, K.M.S. and M.A. ISLAM (2011). Efficacy of citric acid in diet containing low levels of protein and energy on the performance and immunity of broiler (7th International Poultry Show and Seminar. World’s Poultry Science Association-Bangladesh Branch, 25-27 March, 2011, Dhaka, Bangladesh). pp. 318-324.
13. Deepa K., M.R. Purushothaman, P. Vasanthakumar and K. Sivakumar (2017). Serum biochemical parameters and meat quality influenced due to supplementation of sodium butyrate in broiler chicken. International Journal of Livestock Research, Vol 7 (8): 108-116.
14. Emami N. K., S. Z. Naeini and C. A. Ruiz-Feria (2013). Growth performance, digestibility, immune response and intestinal morphology of male broilers fed phosphorus deficient diets supplemented with microbial phytase and organic acids. Livestock Science, 157:506-513.
15. Fascina V.B., G.A.M. Pasquali , F.B. Carvalho, E.M. Muro, F. Vercese, M.M. Aoyagi, A.C. Pezzato, E. Gonzales, and J.R. Sartori (2017). Effects of phytogenic additives and organic acids, alone or in combination, on the performance, intestinal quality and immune responses of broiler chickens. Brazilian J. Poult. Sci., ISSN 1516-635X 19 (3):497-508.
16. Ghasemi H.A., H.A. Salamat, I. Hajkhodadadi and A. H. K. Farahani (2014). Effects of dietary organic acid blend supplementation on performance, intestinal morphology and antibody-mediated immunity in broiler chickens. Advances in Applied Agricultural Sciences, 2(10):64-74
17. Ghazalah A.A., A.M. Atta, K. Elkloub, M.E.L. Mustafa, R.F.H. Shata (2011). Effect of dietary supplementation of organic acids on performance, nutrients digestibility and health of broiler chicks. J. Poultry Sci., 10(3):176–184.
18. Haq Z., A. Rastogi, R. K. Sharma and N. Khan (2017). Advances in role of organic acids in poultry nutrition: A review. Journal of Applied and Natural Science, 9 (4): 2152 – 2157.
19. Haque M.N., Islam, K.M.S., M.A. Akbar, M.R. Karim, R. Chowdhury, M. Khatun and B.W. Kemppainen (2010). Effect of dietary citric acid, flavomycin and their combination on the performance, tibia ash and immune status of broiler. Canadian J. Sci., 90(1): 57-63.
20. Hedayati M., M. Manafi, M. Yari and P. Vafaei (2013). Effects of supplementing diets with an acidifier on performance parameters and visceral organ weights of broilers. European Journal of Zoological Research, 2 (6):49-55.
21. Hudha N., M.S. Ali, M.A. Azad, M.M. Hossian, M. Tanjim,  S.C.Bormon,  M.S. Rahman, M.M.  Rahman and A.K. Paul (2010). Effect of acetic acid on growth and meat yield in broilers. International. Journal of Biological Research, 1(4): 31-35.
22. Kamel, E.R. and L.S. Mohamed (2016). Effect of Dietary Supplementation of Probiotics, Prebiotics, Synbiotics, Organic Acids and Enzymes on Productive and Economic Efficiency of Broiler Chicks. Alexandria Journal for Veterinary Sciences, 50(1): 93-96.
23. Kanagaraju P., S. Ratnaprabha and A.V. Omprakash (2017). Effect of organic acid mixture supplementation on production performance and gut health of commercial broilers. Proceedings of Indian Poultry Science Association Conference 28-30 November 2017. PNR- 59.
24. Khan, S.H. and J. Iqbal (2016). Recent advances in the role of organic acids in poultry nutrition. Journal of Applied Animal Research, 44(1), 359–369.
25. Kil D.Y., W.B. Kwon and B.G. Kim (2011). Dietary acidifiers in weanling pig diets: A review. Rev Colomb Cienc Pecu., 24:231-247.
26. Lakshmi K.V., R.A. Rajashekar, S.G. Shyam and Y.N. Reddy (2016). Dietary supplementation of propionic acid, butyric acid or antibiotic on the performance, carcass parameters and immune response in broiler. Indian Journal of Poultry Science, 51(2):159-163.
27. Lohakare D., M.H. Ryu, T.W. Hahn, J.K. Lee and B.J. Chae (2005). Effects of supplemental ascorbic acid on the performance and immunity of commercial broilers. J. Appl. Poultry Res., 14:10–19.
28. Manafi , M. Hedayati, S. Khalaji and M. Kamely (2016). Assessment of a natural, non-antibiotic blend on performance, blood biochemistry, intestinal microflora, and morphology of broilers challenged with Escherichia coli. R. Bras. Zootec., 45(12):745-754.
29. Marin-Flamand , A. Vazquez-Duran and A. Mendez-Albores (2014). Effect of organic acid blends in drinking water on growth performance, blood constituents and immune response of broiler chicken. J. Poult. Sci., 51:144-150.
30. Miles R.D., G.D. Butcher, P.R. Henry and R.C. Littell (2006). Effect of antibiotic growth promoters on broiler performance, intestinal growth parameters and quantitative morphology. Poultry Science, 85(3):476-485.
31. Mroz, Z., (2005). Organic acids as potential alternatives to antibiotic growth promoters for pigs. Pork Prod., 16:169-18.
32. Naveenkumar S., N. Karthikeyan, R. Narendra Babu, P. Veeramani and Sivaramakrishnan (2017). Effect of Organic Acid Salts as an Alternative to Antibiotic Growth Promoters on the Production Performance of Commercial Broiler Chicken. Int.J.Curr.Microbiol.App.Sci., 6(9): 3470-3480.
33. I.E. Manual for diagnostic test and vaccine (1992). Second edition published by office International Disepizootics.
34. Panda A.K., S.V. Rama Rao, M.V.N.L. Raju and G.S. Shyam (2009). Effect of butyric acid on performance, gastrointestinal tract health and carcass characteristics in broiler chickens. Asian-Aust. J. Anim. Sci., 22(7):1026–1031.
35. Paul S.K., G. Halder, M.K. Mondal, G. Samanta (2007). Effect  of organic acid salt on the performance and gut health of broiler chicken. Poultry Sci., 44:389– 395.
36. Pelicano E. R. L., P. A. Souza and H.B.A. Souza (2005). Intestinal mucosa development in broiler chickens fed natural growth promoters. Revista Brasileira de Ciencia Avícola, 7: 221–229.
37. Ragaa N.M., R.M.S. Korany and F.F. Mohamed (2016). Effect of thyme and/or formic acid dietary supplementation on broiler performance and immunity. Agriculture and Agricultural Science Procedia, 10:270-279.
38. Ramigani V.R., J.V. Ramana, R.D. Srinivasa, S. Shakila and J. Suresh (2017). Effects of dietary supplementation of organic acids in combination on performance and carcass traits of broiler chicken. Animal Nutrition and Feed Technology,17(1):181-187.
39. Rout S.K., C.R. Pradhan, R. Rath, N. Panda, B. Panigrahi and P.K. Patil (2016). Influence of probiotics and acidifier supplementation on growth, carcass characteristics and economics of feeding in broilers. Indian Journal of Animal Nutrition, 33(1): 97-101.
40. Salgado-Transito L., J. C. Del, C. Río-García, J.L. Arjona-Román, E. Moreno-Martínez, A. Méndez-Albores (2011) Effect of citric acid supplemented diets on aflatoxin degradation, growth performance, and serum parameters in broiler chickens. Arch. Med. Vet., 43:215-222.
41. Samanta S., S. Haldar and T.K. Ghosh (2010). Comparative efficacy of an organic acid blend and bacitracin methylene disalicylate as growth promoters in broiler chickens: effects on performance, gut histology, and small intestinal milieu. Med. Int. 2010:645–650.
42. Sohail R., M. Saeed, S. Chao, R.N. Soomro, M.A. Arain, I.H.R. Abbasi and M. Yousaf (2015). Comparative Effect of Different Organic Acids (Benzoic, Acetic and Formic) on Growth Performance, Immune Response and Carcass Traits of Broilers. Journal of Animal Production Advance, 5(9): 757-764.
43. Snedecor, G. W. and W. G. Cochran (2002). Statistical Methods, 8th Edition, Oxford and IBH publishing company Calcutta, Bombay and New Delhi.
44. Sultan A., T. Ullah, S. Khan and R.U. Khan (2015). Effect of Organic Acid supplementation on the performance and ileal microflora of broiler during finishing period. Pakistan J. Zool., 47(3): 635-639,
45. Tanzin M., K.M.S. Islam, M.R. Debi and M.R. Islam (2015). Effect of citric acid, herbal feed additive and their combination on the performance of broiler. J. Anim. Sci., 44 (3): 143-150.
46. Tazoe H., Y.Otomo, I.Kaji, R. Tanaka, S.I. Karaki and A. Kuwahara (2008). Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions. Physiol. Pharmacol., 59 (2): 251-262.