rbzRevista Brasileira de ZootecniaR. Bras. Zootec.1516-35981806-9290Sociedade Brasileira de Zootecnia0260710.37496/rbz5520250013Non-ruminantsEffects of acidifiers in the drinking water on performance, serum biochemistry, and digestive tract microbial counts in broilers0000-0002-0139-9872PagnussattHeloisaConceptualization10000-0002-7120-4911ZaccaronGustavoConceptualization10000-0002-9519-4033SantoAlicia DalConceptualization10000-0002-2868-8498ValentiniFernanda Danieli AntoniazziConceptualizationWriting – review & editing10000-0002-9416-4879VogelThainá DaianeConceptualization10000-0001-8794-0166PrestesAlan MirandaData curation10000-0002-2891-7756GirardiniLilian KollingData curation10000-0002-3161-019XTavernariFernando de CastroData curation20000-0002-0814-8726StefaniLenita MouraData curation30000-0001-8547-4149ValentimJean KaiqueWriting – original draft40000-0002-2282-3580CalderanoArele ArlindoProject administrationWriting – original draftWriting – review & editing50000-0002-6175-5939PetrolliTiago GoulartFunding acquisitionInvestigationProject administrationWriting – original draftWriting – review & editing1*Universidade do Oeste de Santa CatarinaPrograma de Pós-Graduação em Sanidade e Produção AnimalXanxerêSCBrasil Universidade do Oeste de Santa Catarina, Programa de Pós-Graduação em Sanidade e Produção Animal, Xanxerê, SC, Brasil.EMBRAPA Suínos e AvesConcórdiaSC Brasil EMBRAPA Suínos e Aves, Concórdia, SC Brasil.Universidade do Estado de Santa CatarinaChapecóSCBrasil Universidade do Estado de Santa Catarina, Chapecó, SC, Brasil.Universidade Federal Rural do Rio de JaneiroInstituto de ZootecniaDepartamento de Produção AnimalSeropédicaRJBrasil Universidade Federal Rural do Rio de Janeiro, Instituto de Zootecnia, Departamento de Produção Animal, Seropédica RJ, Brasil.Universidade Federal de ViçosaDepartamento de ZootecniaViçosaMGBrasil Universidade Federal de Viçosa, Departamento de Zootecnia, Viçosa, MG, Brasil.tiago.petrolli@unoesc.edu.br
Ines Andretta
The authors declare no conflicts of interest.
08072026202655e202500131102202516112025This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.ABSTRACT
This study evaluated the effects of supplementing drinking water with an acidifier blend on growth performance, serum biochemical profile, and gastrointestinal microbial counts of broilers. A mixture containing phosphoric, citric, and tartaric acids was supplied at 0, 60, or 150 mL/1000 L of drinking water during specific production phases. A total of 320 Cobb male broilers were allocated in a completely randomized design with eight replicates of 12 birds each. The acidifier progressively reduced drinking water pH values (5.54, 3.17, and 2.72, respectively). No effects (P>0.05) were observed on final body weight, feed intake, or feed conversion ratio. A quadratic trend (P = 0.087) was observed for total mesophilic bacterial counts in the crop, with the lowest point estimated at 103.87 mL/1000 L, whereas jejunal bacterial counts were not affected. Quadratic responses (P<0.01) were also observed for serum total protein, globulin, cholesterol, and uric acid concentrations, while albumin was not influenced (P>0.05). Supplementation of drinking water with a blend of citric, tartaric, and phosphoric acids at 150 mL/1000 L was safe, did not impair broiler performance, and maintained serum biochemical values within normal ranges. Quadratic responses among the evaluated concentrations (0, 60, and 150 mL/1000 L) suggest a potential inclusion range between 85 and 157 mL/1000 L associated with favorable outcomes. These findings indicate that the evaluated acid blend may contribute to gut health and represents a promising alternative to antibiotic growth promoters.
Keywords:citric acidintestinal healthmicrobiotaphosphoric acidtartaric acidCoordenação de Aperfeiçoamento de Pessoal de Nível Superior001The authors are thankful for the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC), financial code 001, and the company Engenutri Comércio de Aditivos Alimentares Ltda. for providing the acidifier.1. Introduction
Over the last few decades, growth-enhancing antibiotics have been used to ensure greater feed efficiency in broiler chickens. However, the inappropriate use of these antibiotics at subclinical doses has led to the prohibition of their use for this purpose, aiming to reduce potential risks to consumers, thereby aligning with the concept of One Health. Residues of these compounds are frequently detected in products of animal origin, which can cause bacterial cross-resistance, impacting the efficiency of antimicrobial therapy for humans (Parent et al., 2020).
The poultry industry faces a series of sanitary challenges throughout chicken production, particularly concerning the intestinal health of birds. Gastrointestinal disorders, bacterial and parasitic infections, and the extensive use of antibiotics all negatively contribute to the microbial balance in the gastrointestinal tract of birds (Murshed et al., 2024). In this context, acidifiers, when added via drinking water, can help prevent gastrointestinal diseases (Adil et al., 2010) and boost immune system function, which results in improved growth performance and digestibility (Yadav and Jha, 2019).
Organic acidifiers provide energy, while inorganic acidifiers, such as phosphoric acid, offer nutrients such as phosphorus (Viola and Vieira, 2007), which play a crucial role in improving animal nutrition. Citric and tartaric acids, in particular, contribute to the integrity and stability of the intestinal microbiota by reducing pathogenic agents in the organism, which helps to decrease the occurrence of sanitary problems (Ángel-Isaza et al., 2019). These effects ultimately lead to improved bird performance, and both citric and tartaric acids are also safe for human health, making them potential substitutes for growth-promoting antibiotics (Ángel-Isaza et al., 2019; Saleem et al., 2020).
Phosphoric acid, in addition to its bactericidal effects on Enterococcus faecalis, has been shown to be more effective than citric acid (Arias-Moliz et al., 2008). It is one of the most commonly used acids in drinking water, not only for acidifying but also because it provides phosphorus for animal nutrition (Gao et al., 2021). Phosphoric acid is easily dissolved in water due to its ability to control pH (Lee et al., 2018) and remains stable at high temperatures with low volatility (Murahashi, 2009). Typically, it is found in an aqueous solution, which is colorless and can be corrosive depending on its dosage (Speight, 2017).
The use of acidifiers in the gastrointestinal tract lowers the pH, leading to physiological and morphological modifications that reduce the pathogenic microbial load, which in turn exert positive effects on the site’s physiology and morphology, ultimately improving animal performance and immune function (Dittoe et al., 2018). Phosphoric acid specifically causes an immediate pH reduction in an aqueous medium, which helps it dissociate easily, thereby losing its main function of acidifying the bacterial cytoplasm. However, it still exerts beneficial effects by acidifying the intestinal lumen.
The hypothesis of this research is that supplementation of acidifiers through drinking water can reduce the microbial load in the digestive tract of broiler chickens without compromising the birds’ performance and health. Therefore, the objective of this study was to evaluate the effect of the inclusion of a liquid blend of citric, tartaric, and phosphoric acids broiler performance, total mesophilic microbial counts in the crop and jejunum, and serum biochemical parameters in broiler chickens.
2. Material and methods2.1. Animals, experimental design, and water treatment
This study was approved by the university’s animal ethics committee under protocol number 03/2021.
This study was carried out at UNOESC - Poultry Sector facilities (Xanxerê city, SC, Brazil, Latitude: −26.836432730209985; Longitude: −52.40740389021019), using 320 male broiler chickens of the COBB lineage distributed on the first day of age in a completely randomized experimental design comprising three treatments (control, 60 mL and 150 mL of acidifier per 1000 L of drinking water) with eight repetitions each and 12 animals per experimental unit.
The animals were housed in 2 m2 pens and raised according to the lineage manual, using shaving litter reused for five consecutive flocks, receiving feed (Table 1) in tubular feeders and nipple drinkers with water available ad libitum throughout the experimental period. The blends containing citric (5 – 10%), tartaric (5 – 10%), and phosphoric (60 – 80%) acids were added to nonchlorinated well water from 19 to 22 days, 26 to 28 days and from 33 until the end of the experiment (42 days of life). The broilers were exposed to a light cycle of 16 hours of light and 8 hours of darkness, with a light intensity of 20 lux during light hours, in accordance with the animal welfare guidelines (Directive 2007/43/EC).
Feed and nutritional composition of each reference diet of the experiment according to the rearing phase
2 Mineral supplement (per kg of product): iron - 100 g; cobalt - 2.0 g.
The birds were fed a balanced diet according to the nutritional guidelines recommended by Rostagno et al. (2017). The rations were provided ad libitum, with feeding occurring three times per day. The study period lasted from day 1 to 42 d of age, with feeding programs adjusted according to the growth stages of the birds (1-21 and 1-42 d).
2.2. Drinking water pH
The drinking water pH was measured before and after acid dilution and is presented as an average for each treatment. This measurement was performed using a calibrated digital pH meter (Model HI 8424, HANNA Instruments®, Barueri, São Paulo, Brazil).
2.3. Performance
All animals were weighed weekly throughout the study to monitor growth and feed conversion ratio. Specifically, the broilers were weighed at 1, 21, and 42 d of age, along with the leftover feed, to determine the final weight (FW), feed intake (FI), and feed conversion ratio (FCR). Additionally, at 42 days of age, the productivity efficiency index (PEI) was calculated using the following formula: PEI = (Body weight × Viability)/(Age at slaughter × FCR).
2.4. Microbial analysis
At 42 d of age, one bird per experimental unit was humanely euthanized according to the guidelines of the National Council for the Control of Animal Experimentation (CONCEA, Brasil, 2013). The luminal content of the crop and jejunum was aseptically collected for microbial analysis and transported to the laboratory under refrigerated conditions. Samples were processed using the pour plate technique (Silva et al., 2010) using plate count agar (PCA, Oxoid). Briefly, aliquots of the luminal content were homogenized and serially diluted (up to 10⁻⁴) in sterile saline solution (0.85%). From each dilution, 1 mL of inoculum was plated in duplicate. After incubation at 37 °C for 48 h, colonies were counted on plates containing 30 to 300 colonies, and the results were expressed as colony-forming units per gram of digesta (CFU g⁻1).
2.5. Serum biochemical parameters
Blood samples (1 mL per animal at 42 d of age) were obtained from the brachial vein. Serum samples were obtained by centrifuging the blood at 3000 rpm and subsequently stored at −20 °C for analysis of total protein, albumin, globulin, cholesterol, and uric acid concentrations.
Biochemical analyses were carried out using specific commercial kits (Gold Analisa Diagnóstica Ltda., Belo Horizonte, MG, Brazil) for each parameter, which were measured using a Bioplus 2000 semi-automatic analyzer (Bioplus Produtos para Laboratórios Ltda., Barueri, SP, Brazil) at the Biochemistry Laboratory of the Department of Animal Science, State University of Santa Catarina (UDESC, Chapecó, SC, Brazil). Globulin levels were determined as the difference between total protein and albumin, and the results were expressed in g/dL of blood.
2.6. Statistical analysis
Experimental results were first assessed for normality using the Shapiro–Wilk test. Data meeting normality assumptions were subjected to analysis of variance (ANOVA) to identify significant differences among treatment groups. When appropriate, linear and quadratic regression analyses were applied to evaluate potential trends across the data. All statistical analyses were performed using R software (version 4.3.1), with significance established at a 0.05 probability level.
The pen was considered the experimental unit for performance-related variables (final weight, feed intake, feed conversion ratio, and production efficiency index). For serum biochemical parameters and microbiological analyses, one bird per pen was randomly selected, and thus the individual bird was considered the experimental unit for these variables.
Yij=μ+βi+εij,
in which Yij represents the dependent variables evaluated, such as final weight, feed intake, feed conversion, production efficiency index, total microbial count in the digestive tract (harvest and jejunum), and serum biochemical parameters (total protein, albumin, globulin, cholesterol, and uric acid); μ is the overall mean; βi is the fixed effect of the treatment, represented by the different acidifier dosages (independent variable: 0, 60, or 150 mL/1000 L of water); εij is the random experimental error associated with each observation.
3. Results3.1. Drinking water pH
The average pH of the water used in the control group (without acid addition) was 5.54, while the pH values for the 60 mL/1000 L and 150 mL/1000 L acid treatments were 3.17 and 2.72, respectively.
3.2. Performance
The final body weight, feed intake (FI), feed conversion ratio (FCR), and productive efficiency index (PEI) were not affected (P>0.05) by the addition of liquid acids (Table 2).
Performance of chicken broilers supplemented with acidifiers by drinking water
Variable
Control
60 mL/1000 L
150 mL/1000 L
Linear effect
Quadratic effect
CV (%)
SEM
1-21 days
Weight (g)
969
1025
989
0.763
0.447
7.03
31.391
Feed intake (g)
1301
1342
1346
0.423
0.641
6.84
37.192
FCR
1.41
1.37
1.43
0.482
0.233
3.94
0.0233
1-42 days
Weight (g)
3184
3268
3186
0.992
0.578
5.50
63.794
Feed intake (g)
4958
5042
4912
0.779
0.828
6.43
150.962
FCR
1.59
1.56
1.57
0.722
0.890
6.87
0.0413
PEI
459
474
456
0.863
0.821
13.57
22.168
FCR - feed conversion ratio; PEI - productive efficiency index; SEM - standard error of the mean.
3.3. Microbial analysis
In the control group (without acid addition), microbial counts of total mesophiles in both the crop and jejunum did not differ significantly (P>0.05; Figure 1). Comparatively, the birds that received acid treatments at 60 and 150 mL/1000 L exhibited similar microbial counts in the crop and jejunum, with a trend towards a quadratic reduction in microbial counts in the crop (P = 0.087). The quadratic curve reached its minimum point at a dosage of 103.87 mL/1000 L.
Linear and quadratic regressions for total mesophilic bacterial counts in the crop and jejunum of 42-day-old broiler chickens receiving acidifiers via drinking water.
(A) Crop – linear regression; (B) Crop – quadratic regression; (C) Jejunum – linear regression; (D) Jejunum – quadratic regression.X-axis: Acidifier dose (mL/1000 L of water). Y-axis: Mesophilic bacterial count (log CFU/mL).
3.4. Serum biochemical parameters
The serum biochemical profile of the birds indicated a quadratic effect of acidifier doses on total protein (P = 0.003), globulins (P = 0.001), cholesterol (P = 0.005), and uric acid (P<0.001) concentrations (Table 3). Additionally, a linear effect was observed for serum cholesterol (P = 0.001) and uric acid concentrations (P<0.001) as the acidifier doses increased. No effects were found on serum albumin (ALB) concentration with acid supplementation at different doses (P>0.05). The optimal acidifier concentrations, derived from the quadratic regression, were 86.84 mL/1000 L, 85.56 mL/1000 L, 157.20 mL/1000 L, and 125.16 mL/1000 L.
Serum biochemical parameters of chicken broilers supplemented with acidifiers by drinking water at 42 days of age
The addition of liquid acids to drinking water demonstrated significant effects on reducing the microbial load in the upper digestive tract of birds without compromising productive performance (Ricke, 2019; Bourassa et al., 2018). This effect can be attributed to the mechanism of action of acids on the intestinal microbiota. As described by (Pickler et al., 2012), the antimicrobial action of acidifiers in the gastrointestinal tract depends on factors such as age, pH, oxygen level, acid concentration, site of action, and microbiome composition in each section of the tract. The effectiveness of acidifiers administered intermittently aligns with findings from similar studies, suggesting that continuous use throughout the production period may not be justified (Hamid et al., 2018).
According to Pickler et al. (2012), citric and tartaric acids have antimicrobial functions and can exist in either dissociated or non-dissociated forms (Russell, 1992). When combined with phosphoric acid, they help improve the intestinal microbiota when used in drinking water (Zhang et al., 2022). In the non-dissociated form, these acids carry protons (H+), which, upon entering the bacterial cytoplasm, cause a decrease in pH, disrupting bacterial metabolism and resulting in bactericidal and bacteriostatic effects (Zhang et al., 2022; Dierick et al., 2002).
In this study, acids were administered in their free form (without encapsulation), which influenced their antimicrobial action primarily in the proximal sections of the digestive tract, with reduced impact on distal sections due to decreased acidification along the tract (Abdel-Fattah et al., 2008). Acidifiers are most effective in the crop, where there is no intrinsic pH control (Józefiak et al., 2010), leading to a greater reduction in bacterial populations. Supporting these findings, Van Bunnik et al. (2012) reported that adding acidifiers via drinking water increased acidity in the gizzard and stomach, thereby reducing bacterial presence throughout the gastrointestinal tract.
In the crop, phosphoric acid may play a key role in lowering pH immediately upon contact with the environment. The absence of endogenous secretions in this organ limits its natural acidification capacity, making it more susceptible to the antimicrobial action of acids (Clavijo and Flórez, 2018). The use of acidified water during the rearing cycle reduces bacterial colonization in the upper gastrointestinal tract and subsequent sections (Hamed and Hassan, 2013; Mahmoud et al., 2012), contributing to a lower intestinal pH that inhibits harmful bacteria and promotes a balanced microbiota (Hamid et al., 2018).
Further studies have reported a reduction in bacterial load in the gizzard and gastrointestinal tract of broilers consuming acidified water (Van Bunnik et al., 2012). These findings suggest that the use of acidifiers in drinking water is safe for animal performance, as it does not compromise weight gain, feed intake, or feed conversion, although it does not enhance these parameters, as reported in other studies (Adhikari et al., 2020; Alçiçek et al., 2004). In contrast, positive performance effects in broilers were observed by Hu et al. (2020), suggesting that the reduced pH and increased proteolytic enzyme activity in the proventriculus improve nutrient digestion and exert bactericidal and bacteriostatic effects on pathogenic intestinal microorganisms. Acidifiers in drinking water may compensate for low endogenous acid production, often linked to metabolic disturbances, while controlling microbial load (Hamid et al., 2018).
At high doses, acids can limit water intake and even cause lesions in sensitive organs such as the tongue, esophagus, crop, gizzard, and proventriculus (Sugiharto et al., 2020). However, in this study, the dosages used were below commonly reported levels (200 mL/1000 L), and no lesions were observed at necropsy after 42 d. Mantzios et al. (2023) observed ulcers and lesions in the oral mucosa and esophagus with higher doses, along with reduced performance.
Reducing microbial load in broilers intended for slaughter is an important and ongoing challenge, as contamination increases carcass condemnation rates and poses a risk to public health (Peh et al., 2020). Pathogens such as Campylobacter spp. can colonize birds asymptomatically but may contaminate carcasses and cause disease in humans.
In this experiment, total mesophilic bacterial counts in the crop showed greater reductions in groups receiving acidifiers compared with the control, demonstrating the antimicrobial efficacy of acids in the initial sections of the gastrointestinal tract. Citric acid, for example, reduced the pH to levels around 4.0, creating an environment that is unfavorable for pathogenic bacteria (Laury et al., 2009; Dan et al., 2007).
The water used came from an artesian well with a natural pH of 5.54, and the greatest microbial reduction was observed in group 2, with a final pH of 3.17 (60 mL/1000 L). Most pathogenic bacteria do not survive at pH values below 4.0, reinforcing the recommendation that acids achieve this range.
Although the primary goal is to eliminate harmful bacteria, some beneficial bacteria may also be affected, which should be minimized to preserve the beneficial microbiota (Ricke et al., 2020). The intestinal microbiota is essential for metabolism, productivity, and immunity in birds, as it competes with pathogens and exerts bactericidal and bacteriostatic effects (Clavijo and Flórez, 2018). The maintenance of local microbiota function is crucial to maximize nutrient absorption and support the biochemical processes of the digestive tract (Clavijo and Flórez, 2018).
Monitoring serum biochemical parameters is important for evaluating the efficacy of additives, as it provides insights into the general metabolic state of the birds. In this study, the serum biochemical profile indicated significant quadratic effects on total protein, globulins, cholesterol, and uric acid due to acid supplementation, with linear effects on serum cholesterol and uric acid. Globulins, key components of the immune system (immunoglobulins), increased with the use of acidifiers, suggesting an enhanced immune response (Campbell, 2004). This increase may also be related to inflammatory processes requiring elevated immunoglobulin production (Khan and Iqbal, 2016). Globulins are essential for immunity and dietary restrictions that reduce globulin levels may lead to immunosuppression (Ghazalah and Ali, 2008).
Total protein levels increased in relation to the albumin-to-globulin ratio (McKnight et al., 2020), with reference values of 3.23 and 3.27 g/dL. The control group showed higher values (3.50 g/dL), influenced by the acidifier dosage, with the highest value observed at 60 mL/1000 L (5.00 g/dL) and a slight decrease at 150 mL/1000 L (4.25 g/dL). Total protein levels increase as chickens age, reflecting changes in phenotype and target tissue molecules (Tóthová et al., 2019; McKnight et al., 2020). While albumin levels remained stable, some studies suggest that acidifiers can reduce renal disorders in chickens (Harr, 2006). Changes in the albumin/globulin (A/G) ratio are age-dependent, being lower at 4 d and higher at 45 d, reflecting a balanced metabolism during growth (Tóthová et al., 2019).
Although cholesterol levels increased, they remained within the ranges described by Dierick et al. (2002) of 71.80 to 130 mg/dL, indicating that acidifier use did not affect liver or bile function, thereby ensuring bird welfare. Cholesterol plays a key role in the serum transport of fat-soluble vitamins (A, D, K, and E), bile acid synthesis, and structural integrity of cell membranes (Ludke and López, 1999).
Uric acid levels, indicative of renal function, were within safe limits, as renal dysfunction would increase concentrations (Wang et al., 2021). At high doses, acidifiers can stimulate renal production of free radicals, potentially damaging renal cells (Ghorbel et al., 2017).
The inclusion of acids in broiler drinking water showed promising results for reducing microbial load in the upper digestive tract without affecting productive performance. The antimicrobial effect of acids, particularly in the non-dissociated form, disrupts bacterial metabolism by depleting cellular energy and ultimately leading to bacterial death. The effectiveness in controlling pathogenic bacteria highlights the practical relevance of this method in the poultry industry, providing a positive perspective for gut health management in broilers.
5. Conclusions
Supplementation of drinking water with a citric, tartaric, and phosphoric acid blend (150 mL/1000 L) was safe, did not affect broiler performance, and maintained serum biochemical values within normal ranges. A trend toward reduced crop bacteria suggests potential gut health benefits, supporting its use as an alternative to antibiotic growth promoters.
Acknowledgments
The authors are thankful for the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/MEC), financial code 001, and the company Engenutri Comércio de Aditivos Alimentares Ltda. for providing the acidifier.
ReferencesAbdel-Fattah, S. A.; El-Sanhoury, M. H.; El-Mednay, N. M. and Abdel-Azeem, F. 2008. Thyroid activity, some blood constituents, organs morphology, and performance of broiler chicks fed supplemental organic acids. International Journal of Poultry Science 7:215-222.Abdel-FattahS. A.El-SanhouryM. H.El-MednayN. M.Abdel-AzeemF.2008Thyroid activity, some blood constituents, organs morphology, and performance of broiler chicks fed supplemental organic acidsInternational Journal of Poultry Science7215222Adhikari, P.; Yadav, S.; Cosby, D. E.; Cox, N. A.; Jendza, J. A. and Kim, W. K. 2020. Research Note: Effect of organic acid mixture on growth performance and Salmonella Typhimurium colonization in broiler chickens. Poultry Science 99:2645-2649. https://doi.org/10.1016/j.psj.2019.12.037AdhikariP.YadavS.CosbyD. E.CoxN. A.JendzaJ. A.KimW. K.2020Research Note: Effect of organic acid mixture on growth performance and Salmonella Typhimurium colonization in broiler chickensPoultry Science9926452649https://doi.org/10.1016/j.psj.2019.12.037Adil, S.; Banday, T.; Bhat, G. A.; Mir, M. S. and Rehman, M. 2010. Effect of dietary supplementation of organic acids on performance, intestinal histomorphology, and serum biochemistry of broiler chicken. Veterinary Medicine International 2010:479485. https://doi.org/10.4061/2010/479485AdilS.BandayT.BhatG. A.MirM. S.RehmanM.2010Effect of dietary supplementation of organic acids on performance, intestinal histomorphology, and serum biochemistry of broiler chickenVeterinary Medicine International2010479485https://doi.org/10.4061/2010/479485Alçiçek, A.; Bozkut, M. and Çabuk, M. 2004. The effects of a mixture of herbal essential oil, an organic acid, or a probiotic on broiler performance. South African Journal of Animal Science 34:217-222.AlçiçekA.BozkutM.ÇabukM.2004The effects of a mixture of herbal essential oil, an organic acid, or a probiotic on broiler performanceSouth African Journal of Animal Science34217222Ángel-Isaza, J.; Mesa-Salgado, N. and Narvaez-Solarte, W. 2019. Organic acids, an alternative in poultry nutrition: a review. CES Medicina Veterinaria y Zootecnia 14:45-58. https://doi.org/10.21615/cesmvz.14.2.4Ángel-IsazaJ.Mesa-SalgadoN.Narvaez-SolarteW.2019Organic acids, an alternative in poultry nutrition: a reviewCES Medicina Veterinaria y Zootecnia144558https://doi.org/10.21615/cesmvz.14.2.4Arias-Moliz, M. T.; Ferrer-Luque, C. M.; Espigares-Rodríguez, E.; Liébana-Ureña, J. and Espigares-García, M. 2008. Bactericidal activity of phosphoric acid, citric acid, and EDTA solutions against Enterococcus faecalis. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontoics 106:e84-e89. https://doi.org/10.1016/j.tripleo.2008.04.002Arias-MolizM. T.Ferrer-LuqueC. M.Espigares-RodríguezE.Liébana-UreñaJ.Espigares-GarcíaM.2008Bactericidal activity of phosphoric acid, citric acid, and EDTA solutions against Enterococcus faecalisOral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontoics106e84e89https://doi.org/10.1016/j.tripleo.2008.04.002Bourassa, D. V.; Wilson, K. M.; Ritz, C. R.; Kiepper, B. K. and Buhr, R. J. 2018. Evaluation of the addition of organic acids in the feed and/or water for broilers and the subsequent recovery of Salmonella Typhimurium from litter and ceca. Poultry Science 97:64-73. https://doi.org/10.3382/ps/pex289BourassaD. V.WilsonK. M.RitzC. R.KiepperB. K.BuhrR. J.2018Evaluation of the addition of organic acids in the feed and/or water for broilers and the subsequent recovery of Salmonella Typhimurium from litter and cecaPoultry Science976473https://doi.org/10.3382/ps/pex289Brasil. Ministério da Ciência, Tecnologia e Inovação. 2013. Diretrizes da prática de eutanásia do CONCEA. MCTI, Brasília.BrasilMinistério da Ciência, Tecnologia e Inovação2013Diretrizes da prática de eutanásia do CONCEAMCTIBrasíliaCampbell, T. W. 2004. Clinical chemistry of birds. p.479-492. In: Thrall, M. A.; Baker, D. C.; Campbell, T. W.; DeNicola, D.; Fettman, M. J.; Lassen, E. D.; Rebar, A. and Weiser, G. (eds.). Veterinary hematology and clinical chemistry. Lippincott, Williams & Wilkins, Philadelphia.CampbellT. W.2004Clinical chemistry of birds479492ThrallM. A.BakerD. C.CampbellT. W.DeNicolaD.FettmanM. J.LassenE. D.RebarA.WeiserG.edsVeterinary hematology and clinical chemistryLippincott, Williams & WilkinsPhiladelphiaClavijo, V. and Flórez, M. J. V. 2018. The gastrointestinal microbiome and its association with the control of pathogens in broiler chicken production: A review. Poultry Science 97:1006-1021. https://doi.org/10.3382/ps/pex359ClavijoV.FlórezM. J. V.2018The gastrointestinal microbiome and its association with the control of pathogens in broiler chicken production: A reviewPoultry Science9710061021https://doi.org/10.3382/ps/pex359Dan, S. M.; Mihaiu, M.; Rotaru, O. and Dalea, I. 2007. Microbial changes on the surface of pork carcasses due to lactic and acetic acids decontamination. USAMV-CN 64:403-408.DanS. M.MihaiuM.RotaruO.DaleaI.2007Microbial changes on the surface of pork carcasses due to lactic and acetic acids decontaminationUSAMV-CN64403408Dierick, N. A.; Decuypere, J. A.; Molly, K.; Van Beek, E. and Vanderbeke, E. 2002. The combined use of triacylglycerols containing medium-chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition: I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of piglets. Livestock Production Science 75:129-142. https://doi.org/10.1016/S0301-6226 (01)00303-7DierickN. A.DecuypereJ. A.MollyK.Van BeekE.VanderbekeE.2002The combined use of triacylglycerols containing medium-chain fatty acids (MCFAs) and exogenous lipolytic enzymes as an alternative for nutritional antibiotics in piglet nutrition: I. In vitro screening of the release of MCFAs from selected fat sources by selected exogenous lipolytic enzymes under simulated pig gastric conditions and their effects on the gut flora of pigletsLivestock Production Science75129142https://doi.org/10.1016/S0301-6226 (01)00303-7Dittoe, D. K.; Ricke, S. C. and Kiess, A. S. 2018. Organic acids and potential for modifying the avian gastrointestinal tract and reducing pathogens and disease. Frontiers in Veterinary Science 5:216. https://doi.org/10.3389/fvets.2018.00216DittoeD. K.RickeS. C.KiessA. S.2018Organic acids and potential for modifying the avian gastrointestinal tract and reducing pathogens and diseaseFrontiers in Veterinary Science5216https://doi.org/10.3389/fvets.2018.00216Gao, C. Q.; Shi, H. Q.; Xie, W. Y.; Zhao, L. H.; Zhang, J. Y.; Ji, C. and Ma, Q. G. 2021. Dietary supplementation with acidifiers improves the growth performance, meat quality, and intestinal health of broiler chickens. Animal Nutrition 7:762-769. https://doi.org/10.1016/j.aninu.2021.01.005GaoC. Q.ShiH. Q.XieW. Y.ZhaoL. H.ZhangJ. Y.JiC.MaQ. G.2021Dietary supplementation with acidifiers improves the growth performance, meat quality, and intestinal health of broiler chickensAnimal Nutrition7762769https://doi.org/10.1016/j.aninu.2021.01.005Ghazalah, A. A. and Ali, A. M. 2008. Rosemary leaves as a dietary supplement for growth in broiler chickens. International Journal of Poultry Science 7:234-239.GhazalahA. A.AliA. M.2008Rosemary leaves as a dietary supplement for growth in broiler chickensInternational Journal of Poultry Science7234239Ghorbel, I.; Elwej, A.; Fendri, N.; Mnif, H.; Jamoussi, K.; Boudawara, T.; Grati Kamoun, N. and Zeghal, N. 2017. Olive oil abrogates acrylamide-induced nephrotoxicity by modulating biochemical and histological changes in rats. Renal Failure 39:236-245. https://doi.org/10.1080/0886022X.2016.1256320GhorbelI.ElwejA.FendriN.MnifH.JamoussiK.BoudawaraT.Grati KamounN.ZeghalN.2017Olive oil abrogates acrylamide-induced nephrotoxicity by modulating biochemical and histological changes in ratsRenal Failure39236245https://doi.org/10.1080/0886022X.2016.1256320Hamed, D. M. and Hassan, A. M. 2013. Acids supplementation to drinking water and their effects on Japanese quails experimentally challenged with Salmonella Enteritidis. Research in Zoology 3:15-22.HamedD. M.HassanA. M.2013Acids supplementation to drinking water and their effects on Japanese quails experimentally challenged with Salmonella EnteritidisResearch in Zoology31522Hamid, H.; Shi, H. Q.; Ma, G. Y.; Fan, Y.; Li, W. X.; Zhao, L. H.; Zhang, J. Y.; Ji, C. and Ma, Q. G. 2018. Influence of acidified drinking water on growth performance and gastrointestinal function of broilers. Poultry Science 97:3601-3609. https://doi.org/10.3382/ps/pey212HamidH.ShiH. Q.MaG. Y.FanY.LiW. X.ZhaoL. H.ZhangJ. Y.JiC.MaQ. G.2018Influence of acidified drinking water on growth performance and gastrointestinal function of broilersPoultry Science9736013609https://doi.org/10.3382/ps/pey212Harr, K. E. 2006. Diagnostic value of biochemistry. p.611-630. In: Harrison, G. J. and Lightfoot, T. L. (eds.). Clinical avian medicine. v. 2. Spix, Palm Beach.HarrK. E.2006Diagnostic value of biochemistry611630HarrisonG. J.LightfootT. L.edsClinical avian medicine2SpixPalm BeachHu, Y.; Wang, L.; Shao, D.; Wang, Q.; Wu, Y.; Han, Y. and Shi, S. 2020. Selectively and reshaped early dominant microbial community in the cecum with similar proportions and better homogenization and species diversity due to organic acids as AGP alternatives mediate their effects on broilers' growth. Frontiers in Microbiology 10:2948. https://doi.org/10.3389/fmicb.2019.02948HuY.WangL.ShaoD.WangQ.WuY.HanY.ShiS.2020Selectively and reshaped early dominant microbial community in the cecum with similar proportions and better homogenization and species diversity due to organic acids as AGP alternatives mediate their effects on broilers' growthFrontiers in Microbiology102948https://doi.org/10.3389/fmicb.2019.02948Józefiak, D.; Kaczmarek, S. and Rutkowski, A. 2010. The effects of benzoic acid supplementation on the performance of broiler chickens. Journal of Animal Physiology and Animal Nutrition 94:29-34. https://doi.org/10.1111/j.1439-0396.2008.00875.xJózefiakD.KaczmarekS.RutkowskiA.2010The effects of benzoic acid supplementation on the performance of broiler chickensJournal of Animal Physiology and Animal Nutrition942934https://doi.org/10.1111/j.1439-0396.2008.00875.xKhan, S. H. and Iqbal, J. 2016. Recent advances in the role of organic acids in poultry nutrition. Journal of Applied Animal Research 44:359-369. https://doi.org/10.1080/09712119.2015.1079527KhanS. H.IqbalJ.2016Recent advances in the role of organic acids in poultry nutritionJournal of Applied Animal Research44359369https://doi.org/10.1080/09712119.2015.1079527Laury, A. M.; Alvarado, M. V.; Nace, G.; Alvarado, C. Z.; Brook, J. C.; Echeverry, A. and Brashears, M. M. 2009. Validation of a lactic acid- and citric acid-based antimicrobial product for the reduction of Escherichia coli O157:H7 and Salmonella on beef tips and whole chicken carcasses. Journal of Food Protection 72:2208-2211.LauryA. M.AlvaradoM. V.NaceG.AlvaradoC. Z.BrookJ. C.EcheverryA.BrashearsM. M.2009Validation of a lactic acid- and citric acid-based antimicrobial product for the reduction of Escherichia coli O157:H7 and Salmonella on beef tips and whole chicken carcassesJournal of Food Protection7222082211Lee, J. H.; Kim, H. H.; Cho, Y. H.; Koo, T. S. and Lee, G. W. 2018. Development and evaluation of raloxifene-hydrochloride-loaded supersaturatable SMEDDS containing an acidifier. Pharmaceutics 10:78. https://doi.org/10.3390/pharmaceutics10030078LeeJ. H.KimH. H.ChoY. H.KooT. S.LeeG. W.2018Development and evaluation of raloxifene-hydrochloride-loaded supersaturatable SMEDDS containing an acidifierPharmaceutics1078https://doi.org/10.3390/pharmaceutics10030078Ludke, M. C. M. M. and López, J. 1999. Colesterol e composição dos ácidos graxos nas dietas para humanos e na carcaça suína. Ciência Rural 29:181-187. https://doi.org/10.1590/S0103-84781999000100033LudkeM. C. M. M.LópezJ.1999Colesterol e composição dos ácidos graxos nas dietas para humanos e na carcaça suínaCiência Rural29181187https://doi.org/10.1590/S0103-84781999000100033Mahmoud, M. M. A.; Hasssan, A. M.; Omnia, A. and Kilany, E. 2012. Effect of drinking water pH on performance, apparent ileal digestibility, and some biochemical parameters of Japanese quail. In: The 3rd Mediterranean Poultry Summit and the 6th International Poultry Conference, Alexandria, Egypt.MahmoudM. M. A.HasssanA. M.OmniaA.KilanyE.2012Effect of drinking water pH on performance, apparent ileal digestibility, and some biochemical parameters of Japanese quailThe 3rd Mediterranean Poultry Summit and the 6th International Poultry ConferenceAlexandria, EgyptMantzios, T.; Tsiouris, V.; Papadopoulos, G. A.; Economou, V.; Petridou, E.; Brellou, G. D.; Giannenas, I.; Biliaderis, C. G.; Kiskinis, K. and Fortomaris, P. 2023. Investigation of the effect of three commercial water acidifiers on the performance, gut health, and Campylobacter jejuni colonization in experimentally challenged broiler chicks. Animals 13:2037. https://doi.org/10.3390/ani13122037MantziosT.TsiourisV.PapadopoulosG. A.EconomouV.PetridouE.BrellouG. D.GiannenasI.BiliaderisC. G.KiskinisK.FortomarisP.2023Investigation of the effect of three commercial water acidifiers on the performance, gut health, and Campylobacter jejuni colonization in experimentally challenged broiler chicksAnimals132037https://doi.org/10.3390/ani13122037McKnight, L. L.; Page, G. and Han, Y. 2020. Effect of replacing in-feed antibiotics with synergistic organic acids, with or without trace mineral and/or water acidification, on growth performance and health of broiler chickens under a Clostridium perfringens type A challenge. Avian Diseases 64:374-378. https://doi.org/10.1637/aviandiseases-D-19-00115McKnightL. L.PageG.HanY.2020Effect of replacing in-feed antibiotics with synergistic organic acids, with or without trace mineral and/or water acidification, on growth performance and health of broiler chickens under a Clostridium perfringens type A challengeAvian Diseases64374378https://doi.org/10.1637/aviandiseases-D-19-00115Murahashi, T. 2009. Phosphoric acid fuel cells | Electrolytes. p.564-567. In: Garche, J. (ed.). Encyclopedia of Electrochemical Power Sources. Elsevier. https://doi.org/10.1016/B978-044452745-5.00278-1MurahashiT.2009Phosphoric acid fuel cells | Electrolytes564567GarcheJ.edEncyclopedia of Electrochemical Power SourcesElsevierhttps://doi.org/10.1016/B978-044452745-5.00278-1Murshed, M.; Abudabos, A. M. and Qaid, M. M. 2024. Effects of feeding eubiotics as antibiotic substitutes on growth performance, intestinal histomorphology and microbiology of broilers. Italian Journal of Animal Science 23:65-75. https://doi.org/10.1080/1828051X.2023.2290192MurshedM.AbudabosA. M.QaidM. M.2024Effects of feeding eubiotics as antibiotic substitutes on growth performance, intestinal histomorphology and microbiology of broilersItalian Journal of Animal Science236575https://doi.org/10.1080/1828051X.2023.2290192Parent, E.; Archambault, M.; Moore, R. J. and Boulianne, M. 2020. Impacts of antibiotic reduction strategies on zootechnical performances, health control, and Eimeria spp. excretion compared with conventional antibiotic programs in commercial broiler chicken flocks. Poultry Science 99:4303-4313. https://doi.org/10.1016/j.psj.2020.05.037ParentE.ArchambaultM.MooreR. J.BoulianneM.2020Impacts of antibiotic reduction strategies on zootechnical performances, health control, and Eimeria spp. excretion compared with conventional antibiotic programs in commercial broiler chicken flocksPoultry Science9943034313https://doi.org/10.1016/j.psj.2020.05.037Peh, E.; Kittler, S.; Reich, F. and Kehrenberg, C. 2020. Antimicrobial activity of organic acids against Campylobacter spp. and development of combinations-A synergistic effect? PLoS One 15:e0239312. https://doi.org/10.1371/journal.pone.0239312PehE.KittlerS.ReichF.KehrenbergC.2020Antimicrobial activity of organic acids against Campylobacter spp. and development of combinations-A synergistic effect?PLoS One15e0239312https://doi.org/10.1371/journal.pone.0239312Pickler, L.; Hayashi, R. M.; Lourenço, M. C.; Miglino, L. B.; Caron, L. F.; Beirão, B. C. B.; Silva, A. V. F. and Santin, E. 2012. Avaliação microbiológica, histológica e imunológica de frangos de corte desafiados com Salmonella Enteritidis e Minnesota e tratados com ácidos orgânicos. Pesquisa Veterinária Brasileira 32:27-36.PicklerL.HayashiR. M.LourençoM. C.MiglinoL. B.CaronL. F.BeirãoB. C. B.SilvaA. V. F.SantinE.2012Avaliação microbiológica, histológica e imunológica de frangos de corte desafiados com Salmonella Enteritidis e Minnesota e tratados com ácidos orgânicosPesquisa Veterinária Brasileira322736Ricke, S. C. (ed.) 2019. Improving gut health in poultry. Burleigh Dodds Science Publishing.RickeS. C.ed2019Improving gut health in poultryBurleigh Dodds Science PublishingRicke, S. C.; Dittoe, D. K. and Richardson, K. E. 2020. Formic acid as an antimicrobial for poultry production: A review. Frontiers in Veterinary Science 7:563. https://doi.org/10.3389/fvets.2020.00563RickeS. C.DittoeD. K.RichardsonK. E.2020Formic acid as an antimicrobial for poultry production: A reviewFrontiers in Veterinary Science7563https://doi.org/10.3389/fvets.2020.00563Rostagno, H. S.; Albino, L. T.; Hannas, H. I.; Donzele, J. L.; Sakomura, N. K.; Perazzo, F. G.; Saraiva, A.; Abreu, M. L. T.; Rodrigues, P.B.; Oliveira, R. F. M.; Barreto, S. L. T. and Brito, C. O. 2017. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais. 4.ed. Departamento de Zootecnia, Universidade Federal de Viçosa, Viçosa, MG.RostagnoH. S.AlbinoL. T.HannasH. I.DonzeleJ. L.SakomuraN. K.PerazzoF. G.SaraivaA.AbreuM. L. T.RodriguesP.B.OliveiraR. F. M.BarretoS. L. T.BritoC. O.2017Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais4Departamento de Zootecnia, Universidade Federal de ViçosaViçosa, MGRussell, J. B. 1992. Another explanation for the toxicity of fermentation acids at low pH: Anion accumulation versus uncoupling. Journal of Applied Bacteriology 73:363-370. https://doi.org/10.1111/j.1365-2672.1992.tb04990.xRussellJ. B.1992Another explanation for the toxicity of fermentation acids at low pH: Anion accumulation versus uncouplingJournal of Applied Bacteriology73363370https://doi.org/10.1111/j.1365-2672.1992.tb04990.xSaleem, S. K.; Rahman, A.; Pasha, T. N.; Mahmud, A. and Hayat, Z. 2020. Effects of dietary organic acids on performance, cecal microbiota, and gut morphology in broilers. Tropical Animal Health and Production 52:3589-3596. https://doi.org/10.1007/s11250-020-02396-2SaleemS. K.RahmanA.PashaT. N.MahmudA.HayatZ.2020Effects of dietary organic acids on performance, cecal microbiota, and gut morphology in broilersTropical Animal Health and Production5235893596https://doi.org/10.1007/s11250-020-02396-2Silva, N.; Junqueira, V. C. A.; Silveira, N. F. A.; Taniwaki, M. H.; Santos, R. F. S. and Gomes, R. A. R. 2010. Manual de métodos de análise microbiológica de alimentos e água. Varela, São Paulo.SilvaN.JunqueiraV. C. A.SilveiraN. F. A.TaniwakiM. H.SantosR. F. S.GomesR. A. R.2010Manual de métodos de análise microbiológica de alimentos e águaVarelaSão PauloSpeight, J. G. 2017. Industrial inorganic chemistry. p.111-169. In: Speight, J. G. (ed.). Environmental inorganic chemistry for engineers. Butterworth-Heinemann. https://doi.org/10.1016/B978-0-12-849891-0.00003-5SpeightJ. G.2017Industrial inorganic chemistry111169SpeightJ. G.edEnvironmental inorganic chemistry for engineersButterworth-Heinemannhttps://doi.org/10.1016/B978-0-12-849891-0.00003-5Sugiharto, S.; Pratama, A. R.; Yudiarti, T.; Wahyuni, H. I.; Widiastuti, E. and Sartono, T. A. 2020. Effect of acidified turmeric and/or black pepper on growth performance and meat quality of broiler chickens. International Journal of Veterinary Science and Medicine 8:85-92. https://doi.org/10.1080/23144599.2020.1830691SugihartoS.PratamaA. R.YudiartiT.WahyuniH. I.WidiastutiE.SartonoT. A.2020Effect of acidified turmeric and/or black pepper on growth performance and meat quality of broiler chickensInternational Journal of Veterinary Science and Medicine88592https://doi.org/10.1080/23144599.2020.1830691Tóthová, C.; Sesztáková, E.; Bielik, B. and Nagy, O. 2019. Changes of total protein and protein fractions in broiler chickens during the fattening period. Veterinary World 12:598-604. https://doi.org/10.14202/vetworld.2019.598-604TóthováC.SesztákováE.BielikB.NagyO.2019Changes of total protein and protein fractions in broiler chickens during the fattening periodVeterinary World12598604https://doi.org/10.14202/vetworld.2019.598-604Van Bunnik, B. A. D.; Katsma, W. E. A.; Wagenaar, J. A.; Jacobs-Reitsma, W. F. and De Jong, M. C. M. 2012. Acidification of drinking water inhibits indirect transmission, but not direct transmission of Campylobacter between broilers. Preventive Veterinary Medicine 105:315-319. https://doi.org/10.1016/j.prevetmed.2012.03.007Van BunnikB. A. D.KatsmaW. E. A.WagenaarJ. A.Jacobs-ReitsmaW. F.De JongM. C. M.2012Acidification of drinking water inhibits indirect transmission, but not direct transmission of Campylobacter between broilersPreventive Veterinary Medicine105315319https://doi.org/10.1016/j.prevetmed.2012.03.007Viola, E. S. and Vieira, S. L. 2007. Suplementação de acidificantes orgânicos e inorgânicos em dietas para frangos de corte: desempenho zootécnico e morfologia intestinal. Revista Brasileira de Zootecnia 36:1097-1104. https://doi.org/10.1590/S1516-35982007000500016ViolaE. S.VieiraS. L.2007Suplementação de acidificantes orgânicos e inorgânicos em dietas para frangos de corte: desempenho zootécnico e morfologia intestinalRevista Brasileira de Zootecnia3610971104https://doi.org/10.1590/S1516-35982007000500016Wang, X.; Han, C.; Cui, Y.; Li, S.; Jin, G.; Shi, W. and Bao, Y. 2021. Florfenicol causes excessive lipid peroxidation and apoptosis induced renal injury in broilers. Ecotoxicology and Environmental Safety 207:111282. https://doi.org/10.1016/j.ecoenv.2020.111282WangX.HanC.CuiY.LiS.JinG.ShiW.BaoY.2021Florfenicol causes excessive lipid peroxidation and apoptosis induced renal injury in broilersEcotoxicology and Environmental Safety207111282https://doi.org/10.1016/j.ecoenv.2020.111282Yadav, S. and Jha, R. 2019. Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultry. Journal of Animal Science and Biotechnology 10:2. https://doi.org/10.1186/s40104-018-0310-9YadavS.JhaR.2019Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultryJournal of Animal Science and Biotechnology102https://doi.org/10.1186/s40104-018-0310-9Zhang, H.; Guo, Y.; Wang, Z.; Wang, Y.; Chen, B.; Du, P.; Zhang, X.; Huang, Y.; Li, P.; Michiels, J. and Chen, W. 2022. Acidification of drinking water improved tibia mass of broilers through the alterations of intestinal barrier and microbiota. Animal Bioscience 35:902-915. https://doi.org/10.5713/ab.21.0455ZhangH.GuoY.WangZ.WangY.ChenB.DuP.ZhangX.HuangY.LiP.MichielsJ.ChenW.2022Acidification of drinking water improved tibia mass of broilers through the alterations of intestinal barrier and microbiotaAnimal Bioscience35902915https://doi.org/10.5713/ab.21.0455