INTRODUCTION

The compounds known as organic acids (OA), used in the livestock industry, are aliphatic carboxylic acids formed by carbon, oxygen and hydrogen, they are also called short chain fatty acids (SCFA) or volatile fatty acids; whose structural chain is composed of less than 7 carbon molecules. These compounds are used as one of the alternatives to the use of growth-promoting antibiotics in the poultry industry (^{Ricke, 2003}; ^{Khan and Iqbal, 2016}). Commercially, OAs such as propionic acid have been used for more than 30 years to reduce bacterial and fungal growth in food, in order to hygienically preserve its quality; as well as preventing and controlling infections by Salmonella spp and E. coli K88 in birds and their derivatives (^{Freitag, 2007}; ^{Emami et al., 2017}). On the other hand, due to the prohibition on antibiotic use in animal feed by regulations in the European community and the United States (^{Smith, 2011}), OAs have proven to be an alternative in f the enteric microbiota modulation and inhibition from intestinal pathogenic bacteria, such as E coli, Salmonella Typhimurium, and Campylobacter coli; in addition to promoting the colonization of beneficial flora such as lactobacilli (^{Roth et al., 2017}; ^{Roth et al., 2019}; ^{Bourassa et al., 2018}; ^{Mortada et al., 2020}).

On the other hand, some studies show the positive impact on the productive parameters, intestinal integrity, immune response and intestinal microbiota (^{Emami et al., 2017}; ^{Polycarpo et al., 2017}; ^{Araujo et al., 2018}; ^{Nguyen et al., 2018}; ^{Adhikari et al., 2020}; ^{Al- Mutairi et al., 2020}), benefits that have initially been attributed to the effect they have on the environment of the gastrointestinal tract (GIT), in the digestion process and as a source of energy; but mainly as protection of the same. OAs are found naturally in GIT of birds; which include lactic acid in a higher proportion in the small intestine; while propionic, acetic and butyric acids are found mainly in the blind bags; this is due to the fermentation process (^{Meimandiopur et al., 2011}; ^{Khan and Iqbal, 2016}; ^{Rhot et al., 2017}). The chemical properties, the pH-lowering effects, and the microbial inhibition effectiveness of an acid depend on its pKa value, which is the pH at which OAs are 50% dissociated. The pKa value defines the power of action that OAs can have; the lower its value, it is considered to have a greater capacity to acidify (^{Freitag, 2007}; ^{Khan and Iqbal, 2016}).

The most accepted theory on the action mechanism in the bacterial growth inhibition by OAs considers their liposolubility in media with acidic pH. A pH close to 4.5 maintains the compound liposolubility, which allows them to penetrate the bacterial cell and dissociate in its cytoplasm, generating a metabolic imbalance that ends with the microorganism death (^{Nakal and Siebert, 2003}; ^{Immerseel et al., 2006}). Propionic acid combined with formic acid when used in food, have shown synergy in the control of Salmonella spp, Campylobacter spp and E. coli (^{Roth et al., 2017}; ^{Emami et al., 2017}; ^{Adhikari et al., 2020}); as well as zootechnical benefits. The inclusion of formic acid in combination with propionic acid via drinking water (^{Nhuyen et al., 2018}; ^{Adhikari et al., 2020}), showed favorable results in the parameters in broilers from 1 to 35 days of age, where included 0.02, 0.03, 0.04, 0.05, and 0.06% organic acids.

However, the information does not mention pH values. Other investigations in chickens used a mixture of organic acids (propionic acid, formic acid and butyric acid), to acidify the water to a pH of 3 to 4.5; The results showed benefits in the productive parameters, intestinal microbiota, immune response, nutrient digestibility and carcass quality (^{Ghulam et al., 2013}; ^{Emami et al., 2017}; ^{Polycarpo et al., 2017}; ^{Araujo et al., 2018}; ^{Nguyen et al., 2018}; ^{Adhikari et al., 2020}; ^{Al-Mutairi et al., 2020}); however, the literature available on the use of these OAs used in drinking water and aimed at demonstrating their benefits on zootechnical performance and intestinal pH is scarce.

The objective of the present work was to evaluate the zootechnical performance of the broiler chicken, to the addition of an OA mixture in the drinking water during its productive life; as well as the digestive pH and morphology of the duodenum, blood pH, resistance to jejunum traction and yellowing of the skin.

MATERIAL AND METHODS

RESULTS

The results of the zootechnical parameters are shown in Table 4. An effect was found (p≤ 0.05) of greater body weight (2.7%), in T2 with respect to the control T1. On the other hand, food consumption decreased (p≤ 0.01) in treatment 3, by 3.1% compared to T1 Control and 2.7%, compared to T2; which improved significantly (p≤ 0.01), in the feeding efficiency values in relation to T1 in 5.9% and in 3.8% to T2 with pH of 4. Water consumption was lower (p≤ 0.01) in the control group (T1), where the OA were not added to the drinking water in relation to the birds that were consuming water with OA (T2 and T3); which represented an increase of 2.23 and 2.42% respectively. There were no effects (p≥0.05) in the percentage of mortality between the evaluated treatments.

The gastrointestinal pH values did not show significant effects (p≥ 0.05), among the treatments studied in the different evaluated segments. As shown in table 5, in general values are seen at the level of the average and/or below, those birds that consumed water with the addition of OA (T2 and T3).

Histological measurements of the duodenum (Table 6) did not show differences (p≥0.05) in the length of villi and in the depth of Lieberkuhn crypts, between the evaluated treatments. In the width of the villi, differences were observed (p≤ 0.01), with the highest values in treatment 3, with differences of 23 and 42 microns, in relation to T1 and T2; This effect influenced the results of the calculated digestive area, with the highest values (p≤ 0.01), the morphology of the Control treatment with pH of 8 and 4.

Blood pH was higher (p≤ 0.02) in T1, in relation to treatments 3 and 2 respectively, which showed lower blood pH; likewise, the jejunum tensile strength showed effects (p≤0.03), among the evaluated treatments; being treatment 1 the one that showed the lowest resistance than treatments 3 and 2 respectively, without showing effects (P≤0.05) among the treatments evaluated in the values of yellow pigmentation in skin, as observed in table 7.

DISCUSSION

The zootechnical benefits obtained with the addition of OA have been verified with different compounds, such as fumaric, formic, acetic and propionic acids, (^{Ghulam et al., 2013}; ^{Broom, 2015}; ^{Emami et al., 2017}; ^{Al-Mutairi et al., 2020}); as well as in the mixture of these; where the formic acid combination with acetic acid and propionates has been shown to have a positive effect on productive performance, intestinal morphology, immune response and on the intestinal microbiota (^{Adil et al., 2011}; ^{Polycarpo et al., 2017}; ^{Araujo et al., 2018}; ^{Nguyen et al., 2018}). Historically, these benefits have been initially associated with the native and pathogenic microflora modulation of the intestinal tract, demonstrated by the effect that OAs have in drinking water and food, to counteract the negative effect of pathogenic bacteria, such as Salmonella spp. , E. coli, Campylobacter spp and Clostridium spp., (^{Immersel et al., 2006}; ^{Emami et al., 2017}; ^{Adhikari et al., 2020}; ^{Bourassa et al., 2018}; ^{Mortada et al., 2020}), through colonization of the digestive tract by Lactobacillus. These are considered to be resistant to acidic pH, having a protective role in the gastro-intestinal tract (^{Jin et al., 1998}). ^{Broom (2015)} mentions that in an acid medium of pH 4.6 the effect of OAs as antibacterials is more effective, based on evaluations carried out in vitro, where they conclude that the minimum inhibitory concentration for OAs may vary with the type of bacteria and acid used. Other in vitro studies with Escherichia coli cultures, (^{Dibner and Buttin, 2002}), add different acids, including hydrochloric, formic, lactic and hydroxy-methionine analog (HMTBa), to acidify the culture medium to pH 4 and pH 7, showed that the antibacterial effect is more efficient with pH 4; in addition to making evident the low microbicidal activity of hydrochloric acid. By using a commercial product that included formic and propionic acid in concentrations of 50 mM formic acid, bacteria could be sensitized by an osmotic process (^{Sánchez et al., 2009}); however, it can be deduced that the pH level is more important as a criterion in the dosage of acids than their molar concentration.

This behavior in chickens of T3, contrasts with the primary role of the aforementioned microbial modulation arguments, which suggests as more important the effect that OAs have on the digestive process, since the birds were not challenged to any pathogens, nor did they present no pathological process during the test period. First, in the digestive process, the medium acidification allows a greater transformation of pepsinogen into pepsin at the proventriculus and gizzard level (^{Cuca et al., 2009}) and protein digestion is more efficient; in the same way, the work at pancreas level is improved by increasing its secretions, and in its case, better activity of some exogenous enzymes, such as phytases and mananasses (^{Rafacz et al., 2005}).

^{Angel et al., (2013)} refer to a work where they analyze the pH water effect on GIT pH, when comparing two levels (8.1 vs. 5.8), where an effect was had on the acidic environment of each one of the intestine segments, and as a consequence the group with acidified water at pH 5.8, positively affects the digestion of dry matter; as well as the apparent ileal phosphorus digestibility. The authors explain that part of the lower digestion at alkaline pH is due to the lower effectiveness of the phytases included in the diet. When the pH rises above 4, their efficiency tends to decrease due to their optimal operating pH and due to the precipitation of phytate-calcium chelates. On the other hand, calcium absorption is favored by acidification of the intestinal environment (by keeping calcium salts in solution), carboxylic acids such as propionic and formic, react with carbonates to form soluble salts in water and carbonic acid ( ^{Brown, 2002}), which favors the growth and strength of the bone system (^{Nourmohammadi, 2013}).

In the present work, a greater resistance in jejunal rupture was observed with acidification in T3 at a pH of 6. The benefits of better mineral absorption have been reported; firstly, calcium for the case of bone tissue and trace minerals such as zinc, copper and magnesium; which play an important role in the connective tissue formation. Without forgetting other functions that OAs play in a natural way in metabolism, as a source of energy to intestinal epithelium cells (^{Boroojeni et al., 2014}; ^{Broom 2015}; ^{Yang et al., 2019}), and their beneficial effects on the immune system of the gastrointestinal tract, such as the activation of lymphocytes and macrophages; as well as a greater development of the thymus and Fabricius bursa (^{Ghulam et al., 2013}; ^{Kim et al., 2013}; ^{Emami et al., 2017}; ^{Al-Mutairi et al., 2020}).

The results of the present study with respect to the intestine histo-morphology, do not show a clear relationship between the differences of each treatment with respect to the evaluated lengths (length, width and crypt), however when analyzing the calculated area of the duodenum, A smaller surface area was appreciated in the group with better zootechnical results (T3), in this regard it is noted that the effect that OAs have on the morphology of the small intestine has been demonstrated by several researchers, most of whom have found a greater length of the villi in different segments of the gastrointestinal tract, with an important correlation with body weight and feeding efficiency (^{Nourmohammadi, 2013}; ^{Emami et al., 2017}; ^{Yang et al., 2019}), however, ^{García et al., 2007}, reported greater height of the villi in the groups added with OA without changes in the calculated area of the villi, but with a significant effect (P <0.01) on efficiency to nutritional and digestibility. It is important to point out that a greater height of the intestinal villi is not synonymous with a greater absorption surface (^{Yamauchi, 2007}); the variations in the width are usually related to cellularity degree, since the greater the width; greater infiltration of inflammatory cells located mainly in the lamina propria (^{Maisonnier et al., 2003}). So in the present study, the birds that were treated with acidified drinking water with a pH of 6, showed a lower calculated area of the villi, which coincides with the zootechnical results of the work of ^{García et al., 2007}; which suggests little cellularity, characteristics of a functional structure and no inflammatory processes. Acidification in drinking water also affected blood pH levels among the evaluated treatments (p <0.02); since the values were lower in the birds that were with OA. The influence that food can have on blood pH has been shown, although there is a mechanism that promotes balance to neutrality. In the case of acids in solution, they allow the passage of hydrogen ions through biological membranes, this phenomenon is based on the mechanisms of digestive absorption and ionic exchanges between the digestive and blood compartments (Sturkie, 2000; ^{Meschy, 2015}). This possibility of the passage of hydrogen ions opens up an alternative in the control of metabolic alkalosis problems caused by birds’ hyperventilation in heat stress states (Sturkie, 2000).

CONCLUSIONS AND IMPLICATIONS

The acidification of drinking water with 0.3L/1000 L of OA (formic acid 31%, propionic acid 19%, ammonium format 26% and ammonium propionate 6%), had a positive effect on the productive behavior in the chicken of fattening; Its beneficial effect lies in primarily facilitating the digestion process, where moderate acidification at a pH of 6 is sufficient to achieve better body weight and feed efficiency. The acidification in the drinking water with OA at 0.03 L/1000 L decreased the blood pH, thus opening an alternative in the control of metabolic alkalosis problems, which can be caused by birds’ hyperventilation in heat stress states.