Introduction

There is growing interest on organic acids and their salts (OA+AS) as an alternative to antibiotic growth promoters in broiler production. However, research examining the potential effects of organic acids has been contradictory; some studies reporting positive effects (Paul et al., 2007; Menconi et al., 2014; Adhikari et al., 2020), and others no significant improvement (Açıkgöz et al., 2011; Araujo et al., 2019). Different combinations of organic acids and their efficacy compared to other additives, such as probiotics and essential oils, have been tested. Organic acids have demonstrated to alter the bacterial cell membrane that, in turn, affects energy pathways (Ricke, 2003). These effects could be beneficial for controlling a broad range of pathogens, including those of importance for the poultry industry, such as Campylobacter, Clostridium, E. coli and Salmonella (Polycarpo et al., 2017). The addition of organic acids to drinking water provided to broilers can be used to improve water quality, as well as control the microbiome population in the upper parts of the intestinal tract and reduce bacterial overload in the intestines. However, the amounts and conditions needed to maximize the beneficial effects of organic acids have not been established (Oviedo, 2006).

Ammonium salts used with the mixture of organic acids function as their vehicles, increasing the antimicrobial effect in the intestine, including the development of intestinal villi (Paul et al., 2007). A blend of formic and propionic acids increased the population of beneficial bacteria, such as Lactobacillus spp. (Nava et al., 2009), which produce lactic acid that provides nutrients for enterocytes and promotes trophic effects on the intestine. Furthermore, organic acids alter the gastrointestinal pH, acidifying the crop, proventriculus and gizzard (Broom, 2015) while also enhance digestive enzymes activity and reduce chelate formation of electrostatically charged molecules in the diet with citric acid (Vieira et al., 2018). Together, these benefits improve nutrient digestion and absorption that translate to increased poultry performance.

The effects of titrating drinking water with organic acids to obtain different pH values and different time periods of inclusion during broiler production have not been fully studied. Therefore, the objective of this study was to evaluate production parameters of broilers offered organic acids and their salts in the drinking water to determine the pH value and the time for supplementation. Intestinal morphology and pH values in the intestinal tract and blood were evaluated to optimize a blend of organic acids (formic acid 31%, propionic acid 19%), and their ammonium salts (ammonium formate 26%, and ammonium propionate 6%) (OA+AS) (Novus International, Mexico) in water for improved broiler performance.

Materials and Methods

Results

At 28 days of age, broilers in the different treatment groups showed statistically significant differences in body weight gain (Table 1). Higher BW were observed for broilers given pH 6 water through 28 and 42 days compared to those receiving pH 4 water through 21 and 28 days of age. On day 35, the group that continuously received pH 6 water had increased BW compared to treatments receiving pH 4 water or the control pH 8 water lacking organic acid. Broilers receiving pH 6 drinking water had increased BW up to day 42 compared to those given control pH 8 water and those for which pH 4 water was discontinued at 21-days-old (p≤0.05).

No significant differences were found in feed consumption among treatments through all phases of evaluation (Table 2).

FCR were not different among treatments through 35-days-old (Table 3). At 42-days-old, the group that continuously received pH 6 water had a decreased FCR relative to the group that received pH 4 water through 21-days-old. However, the similar feed consumption in the groups translated to statistically similar FCRs among the group regardless of the increase in BW observed for some treatments.

Average mortality (3.29%) was not affected by treatments, suggesting the absence of specific challenges during the trial, which was expected based on the experimental conditions.

Water consumption was higher on day 21 for the group that received OA+AS with pH 6, however, at 42 days consumption of drinking water was similar (p≥0.05) between treatments, averaging 9.705 L per chicken. At 28 and 35 days of age, only broilers that continuously received water pH 6 - 0.038 mol had higher water consumption than those receiving the same pH with organic acids mixture for 21 days and those that received drinking water with pH 4 - 0.128 mol, until day 28.

The inclusion of the OA+AS in both pH 4 and 6 on water significantly reduced (p≤0.05) blood pH at 40 days of age compared to the control group with pH 8 (7.89 vs 7.75 on pH 4 and 6). Additionally, broilers receiving water at pH 4 or 6 containing the OA+AS during the first 28 days had higher blood pH at 40 days of age. At 27 days of age, the groups in which OA+AS was added to the water (pH 4 and 6, respectively), had a lower pH in duodenum (6.41, 6.30 and 6.32) and ileum (7.11, 6.94 and 6.95) compared to the control group with pH 8.

Intestinal villi (Table 4) were shorter in broilers in the control group that were given water lacking OA+AS (pH 8) compared to those receiving pH 6 water (p≤0.05). Meanwhile, villi were wider in the control group and in the group receiving pH 4 water than broilers receiving pH 6 water (p≤0.01). OA+AS supplementation increased crypt depth of pH 4 and 6 groups compared to the control group (p≤0.01). The number of villi in 1,000,000 µ² was increased in the pH 6 group compared to those receiving pH 4 or the control (p≤0.01). Meanwhile, there were no significant differences (p≥0.05) in surface area among treatment groups. In a comparison of intestinal segments, higher values were observed in duodenum samples for villi length and width as well as crypt depth compared to samples from the ileum, although the number of villi was higher in ileum compared to duodenum (p≤0.01).

Discussion

The addition of OA+AS to drinking water given to broiler chickens may be beneficial for enhancing growth and could be a viable alternative to the use of other growth promoters like antibiotics. The ammonium salts used with the mixture of organic acids are a complement that facilitate their inclusion in water or feed by not reacting with metals; in this way, when the free organic acids and salts reach the proventriculus, the acidic pH of the gastric juice triggers a reaction that transforms them into free acids and, from this point on, follow the same metabolic route as free organic acids, maintaining a synergistic effect on intestinal health (Paul et al., 2007).

However, the optimal OA+AS needed to maximize growth has not been fully determined. Al-Mutairi et al. (2020) reported that broilers given water containing either 2% formic acid or 2% butyric acid exhibited reduced BW, but the combination of these two organic acids did not negatively impact growth performance. Results of another study indicate that supplying formic and propionic acids in their ammonium salts (formate and propionate), at up to 3% in feed, did not impact broiler performance by 42 days of age (Paul et al., 2007). In the present study, the percentage of OA+AS given in the water was 0.03%, which is substantially lower than that used in previous studies.

Other studies showed that changes in BW are related to the type of organic acid used and the pH of the water obtained by titration. Açıkgöz et al. (2011) and Ao (2005) reported negative responses in BW in broilers given water containing formic and citric acids to yield a pH of 4.5 and 3.26, respectively. In the current study, we used a blend of formic and propionic acids at a molecular concentration of 0.128 and 0.038 moles for pH 4 and 6, respectively, and saw similar positive results to those reported in a study by Eftekhari et al. (2015), which evaluated the effects of the same blend, but did not report the molecular concentration of organic acids.

Vale et al. (2004) examined the effects of including 0.5, 1.0, 1.5 and 2.0% of a mixture of propionic and formic acid in feed, reporting that at 2.0% organic acid growth was negatively impacted, but 0.5, 1.0 and 1.5% had no substantial effect on broiler performance up to 42 days.

In terms of mortality, a meta-analysis of 121 studies by Polycarpo et al. (2017) showed that antibiotics, but not organic acids, demonstrated significantly reduced mortality rates compared to treatments that lacked antibiotic growth promoters. They further found that inclusion of organic acids did not produce substantial differences in mortality relative to control group and diets that included antibiotics, despite the use of organic acids in the field to support flocks under challenging conditions. It is possible that organic acids incorporated in water rather than feed could have different effects on mortality rates in the field, and the type of microorganism under challenge. The water management program could also impact mortality. Highly acidic water can be unpalatable, and thus the pH can affect water consumption (Tabler et al., 2013). However, Ao (2005) reported no effect on consumption of pH 3.26 water containing citric acid. Furthermore, in the present study we observed that OA+AS supplementation had no effect on feed consumption, similar to that reported by Açıkgöz et al. (2010), who supplemented with formic acid and reduced pH from 7.4 to 4.5. Similar results were also reported by Bourassa et al. (2018) with the same organic acid. Pinchasov and Jensen (1989) studied how several molecules such as organic acids - including formic and propionic acid- affected feed consumption, concluding that only propionic and acetic acids tend to depress appetite. Together, these results highlight the importance of adhering to recommendations for each product, not exceeding the recommended dose in water or feed.

In terms of gastrointestinal pH, Al‐Tarazi and Alshawabkeh (2003) found a reduction in pH in the crop and cecum of broilers given water supplemented with formic and propionic acid, whereas Açıkgöz et al. (2011) saw no reduction in crop pH following supplementation of formic acid in the drinking water.

The results of the present study demonstrate that including OA+AS in water could reduce pH in blood, which contributes to the physiological normal status of the bird and could be considered as a tool to mitigate the effects of heat stress (Sandercock et al., 2001).

There were differences in the length, width, crypta and number of villi between treatments and anatomical regions evaluated; however, when analyzing the calculated area, a numerical difference, although no statistically, to a greater surface area (p=0.17) was observed in the group with better performance (pH 6) despite presenting a lower villi width, which could be related to the degree of cellularity, with lower infiltration of inflammatory cells in the lamina propia (Maisonniers-Gomez et al., 2003). This suggests little cellularity, characteristic of a functional structure and no inflammatory processes, coinciding with the results of Arce et al. (2020). However, it is important to point out that a greater height of the intestinal villi is not synonymous with a greater absorption surface (Yamauchi, 2007). Therefore, in the present study, several traits were considered to obtain the absorption area or capacity of the intestinal morphology as a whole.

The results obtained are consistent with previous studies reporting that formic acid alone or in combination with propionic acid in feed was associated with positive effects on intestinal morphometry (García et al., 2007; Senkoylu et al., 2007). This effect has been related to the ability of organic acids to promote beneficial microorganisms in the gastrointestinal tract; however, this was not an objective of this work. These microorganisms produce short-chain fatty acids that promote proliferation and growth of enterocytes (Dibner and Buttin, 2002).

Organic acids and their salts promote intestinal health by acidifying the cytoplasm of pathogens (Ricke, 2003). Additionally, they may have positive effects on digestion by increasing enzyme secretion and improving absorption of dietary minerals (Dibner and Buttin, 2002), which together enhance weight gain.

These findings would explain the growth promoting effect found in the present study for groups that received water with pH 6 up to 42 days of age. Sanitization of water in a holistic approach can decrease microbial concentrations through use of correct cleaning procedures and sanitizers such as chlorine, which is commonly used in field conditions. However, chlorine must be present as hypochlorous ions to act as a strong sanitizer. In this form, chlorine affects membrane permeability of microorganisms, particularly Gram negative bacteria, reducing the pathogenic activity of endotoxins and exotoxins (Rosen and Klebanoff, 1982). Reductions in pH can increase the proportion of bound chlorine, and thus acidification is a useful strategy to reduce the amount of chlorine required for sanitization. Furthermore, a drop in pH with the use of organic acids can stabilize chelating molecules present in feed as phytic acid, which in turns improves the efficiency of phytase, trace minerals and vitamins (Vieira et al., 2018).

Hamid et al. (2018) reported that a discontinuous supply of formic and propionic in water produced the same performance in broilers as a continuous supply for up to 42 days. They concluded that acidification of drinking water from pH 7.8 to 4.2 achieved comparable production results to antibiotic growth promoters, mainly by establishing gastric acidity and reducing total aerobic bacteria count. In the present study, pH 6 water (0.038 moles) given continuously for 42 days resulted in better weight gain with no change in feed consumption and improved FCR. Intestinal morphological results demonstrated a positive trophic effect on intestinal cells in groups given pH 6 water, even relative to pH 4 water, which could probably reflect modulation of the microbiome, since pH 6 would allow selection of beneficial populations that promote intestinal cell growth as demonstrated (Nava et al., 2009), with a blend of formic and propionic acid.

This intestinal cell growth promotes maturation of intestinal cells that produce essential enzymes for digestive processes and nutrient absorption that also improves feed conversion efficiency. Also, a physiological benefit was obtained using pH 6 water through a decrease in blood pH without any effect on water and feed consumption. Water having pH 4-5 is commonly used in the field based on in vitro studies that demonstrated an inhibitory effect on bacterial populations like Campylobacter (Chaveerach et al., 2002), and in an in vivo evaluation of Salmonella (Bourassa et al., 2018). However, under experimental conditions that involve no microbial challenges and reduced stress factors than experienced in commercial conditions, our findings indicate that better results could be obtained with pH 6 water and demonstrate that different recommendations should be followed according to the OA+AS used.

In conclusion, inclusion of OA+AS in drinking water at pH 6 (concentration of 0.038 molar) for 42 days improves body weight gain and FCR in broilers not exposed to challenge conditions. The beneficial effects of the OA+AS mixture on performance are related to improvement in length and number of intestinal villi, as well as in absorption area, and reduction in blood, duodenum and ileum pH.