Formic acid as an antibiotic alternative in broiler diets: effects on growth, carcass characteristics, blood chemistry, and intestinal microbial load

Abstract

This study explored the ability of formic acid (FA) to replace antibiotics in broiler chicken diets. It examined how FA affected the chickens' growth, carcass characteristics, blood chemistry, and gut bacteria. The experiment randomly assigned 300 one-day-old (Ross 308) broiler chicks to 5 groups, each divided into 6 replicates with 10 unsexed chicks. The following were the treatments: 1st group, negative control (NC): only received a basal diet; 2nd group, positive control (PC): received a basal diet supplemented with 0.5 grams of Colistin antibiotic per kilogram of diet; 3rd, 4th, and 5th groups (FA2, FA4, and FA6) these groups received a basal diet along with formic acid added at increasing levels: 2, 4, and 6 Cm³ per kilogram of diet, respectively. Results found no significant differences in live body weight (LBW) or body weight gain (BWG) between treatment groups, except for LBW at one week and BWG at 0 to 1 and 4 to 5 wk of age. No significant variations were found in feed intake (FI) and feed conversion ratio (FCR) among the treatment groups, excluding FI and FCR at 1 to 2 wk of age. The treatments significantly impacted carcass traits, dressing percentage, breast meat, thigh meat, spleen, giblets, blood levels of urea, creatinine, total protein, globulin, and albumin, as well as the activity of enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in chicks fed different diets compared to control groups. The addition of FA to the diet significantly impacted antioxidant levels. Also, the FA2 group had the highest total bacterial count (TBC). However, the FA6 group was the opposite; it had the lowest levels of harmful bacteria, such as E. coli and Coliform. Supplementing broiler diets with formic acid improves blood parameters, antioxidant activity, and gut bacteria counts, with 4.0 cm³ formic acid/kg diet supplementation promoting optimal broiler health and product quality.

INTRODUCTION

With the global population booming, demand for poultry has soared. Producers must meet this demand while ensuring their products' characteristics and safety. In the past, conventional antibiotics were widely used in poultry farming to prevent or treat bacterial infections. However, the overuse and misuse of these drugs have had a serious unintended consequence: the rise of antibiotic-resilient bacteria, which currently presents a serious risk to public health (Abreu et al., 2023; Youssef et al., 2023a,b; Youssef et al., 2024). With the world's population constantly increasing, feeding everyone becomes a challenge. To meet this need, we must ensure our food production systems can keep up (United Nations Department of Economic and Social Affairs, 2022).

Currently, poultry is a top choice for meat eaters worldwide. It's the second most popular European European Union (EU) meat behind pork (EuroStat, 2022). Global meat production has also increased (Richie et al., 2022). Looking at the big picture, the Food and Agriculture Organization (FAO) reports that in 2020, poultry meat accounted for nearly 40% of all meat produced globally (FAO, 2023). This led to a worldwide trend of using intense farming techniques. These methods make diseases more likely to spread, comprising diseases that can jump from animals to humans (zoonosis). This can harm the health of the animals and make them less productive (Nazeer et al., 2021; Richie et al., 2022).

The rising demand for animal products isn't just about food safety worries – it raises concerns about the sustainability and safety of these production systems. Raising animals has a built-in impact on “One Health,” which recognizes the connection between animal, human, and environmental health. This impact includes increased greenhouse gases, contaminated drinking water and the environment, the extent of antibiotic confrontation in bacteria, and the rise of new and old zoonotic infections (Espinosa-Marrón et al., 2022; Singh et al., 2024). Ensuring enough food is produced to nourish everyone on Earth is one of today's biggest hurdles (Sousa et al., 2024).

The crowding of animals in large factory farms and the routine utilization of antibiotics to keep them healthy and prevent meat contamination have created a breeding ground for antimicrobial resistance. This is a major public health threat. To address this, some farmers are using dietary acids as an alternative. These acids come in two main types: organic and inorganic. Organic acids (OAs), like the fatty acids found in many foods, are a common choice for poultry feed. Their characteristic structure, R-COOH, identifies them, but it's significant to observe that not all organic acids are suitable for additives (Muneeb et al., 2024; Rocha and Archer, 2024). Poultry producers appreciate the benefits of short-chain organic acids like formic, acetic, propionic, and butyric acids. These acids are not only good for the birds themselves, but they're also easy to add to their feed.

Further, OAs like lactic, tartaric, and citric acids are sometimes used in poultry diets (Dibner and Buttin, 2002). Recently, supplementing chickens’ diets with OAs has become a popular alternative due to its ability to control harmful bacteria. These OAs can significantly improve a bird's growth, feed efficiency, and immunity to disease. They work by lowering the pH level in the digestive tract, which helps the bird better absorb various nutrients from its feed (Kil et al., 2011). The impact of formic acid (FA) on broiler chick growth performance has been a major area of research for many scientists (Saki et al., 2011; Rashid et al., 2020; Stamilla et al., 2020; Al-Ghamdi, 2023). Several reports have shown that adding formic and propionic acids to chicken feed can decrease Salmonella and E. coli levels in their intestines and droppings (Moharrery and Mahzonieh, 2005; Ragaa et al., 2016; Ricke et al., 2020). The present study hypothesized that using formic acid may be a good substitute for antibiotics in broiler diets. This research investigated whether formic acid could be utilized as a substitute for antibiotics that stimulate the growth of broiler feeds. We examined the impact on the birds' growth, carcass feature, blood chemistry, and gut bacteria, aiming to promote healthy food production.

MATERIALS AND METHODS

Ethical Standards of Scientific Research (No. AY2019-2020/Session 6/2020.01.13) from Tanta University, Egypt guidelines for using and maintaining laboratory animals were followed in terms of animal maintenance and care.

Design, Birds, and Diets

In a fully randomized design trial, 300 one-day-old Ross 308 broiler chicks with comparable initial mean body weights were randomly allocated to 5 groups. Ten unsexed chicks from each group's 6 replicates were used. Five weeks passed during the experiment. The NRC's nutritional criteria were met by the basal diet that was provided to the chicks (1994). All chicks were fed pelleted feed throughout the trial (wk 1–5), as shown in Table 1.

Table 1.

Composition and calculated analysis of the basal diet.

Items	Starter (1–21 d)	Grower (22–35 d)
Ingredients (%)
Yellow corn	54.10	58.7
Soybean meal (44% protein)	34.43	29.78
Corn gluten (60% protein)	5.50	5.50
Limestone	1.08	0.95
Dicalcium phosphate	2.00	1.75
Premix1	0.30	0.30
Salt	0.30	0.30
L-lysine	0.29	0.24
DL-methionine	0.20	0.18
Soybean oil	1.80	2.30
Calculated analysis2
Crude protein %	23.12	21.10
Metabolizable energy (Kcal/kg)	3001	3180
Calcium %	0.99	0.89
Available phosphorus %	0.51	0.46
Potassium %	0.54	0.52

¹Minerals and vitamins premix manufactured by Multi Vita Animal Nutrition® (Tenth of Ramadan City, Sharkia Governorate, Egypt) provides vitamin A 12000 IU, vitamin D3 2500 IU, vitamin E 20 mg, vitamin K3 2 mg, vitamin B1 2 mg, vitamin B2 5 mg, vitamin B6 2 mg, vitamin B12 0.05 ug, niacin 30 mg, biotin 0.05 ug, folic acid 1 mg, pantothenic acid 10 mg, manganese 60 mg, zinc 50 mg, iron 40 mg, copper 10 mg, iodine 0.6 mg, selenium 0.3 mg per 1 kg diet. DL-methionine (manufactured by Evonik Industries, Essen, Germany) and contains 99 % methionine. Lysine = lysine hydrochloride (Evonik Industries) and contains 70 % Lysine.

²Calculated according to NRC (1994).

Two stages of feed were given to the chicks: starter feed for the first three weeks (1-21 days) and grower feed for the following two weeks (22–35 d). All the chicks were kept in the same environment with the same care and hygiene routines. The treatments were as follows: the negative control group (NC) had no additives and is just the basal diet. Positive control group (PC): which obtained the basal diet plus 0.5 grams of Colistin antibiotic per kilogram of feed, formic acid groups (FA), FA2, FA4, and FA6: these groups received the basal diet supplemented with formic acid at levels of 2, 4, and 6 cubic centimeters per kilogram of feed, respectively. The chicks were housed in standard cages measuring 1.0 × 1.0 × 0.5 meters throughout the experiment. They had ad libitum access to both food and fresh water. We also kept them on a consistent light schedule of 23 h, followed by 1 h of darkness.

Measurements

Growth Performance

They were weighed individually each week to track the chicks' growth and determine their live weight (LBW) and body weight gain (BWG). Their feed conversion ratio (FCR) and average daily feed intake (FI) were also determined. Six chicks from every group were chosen at random for carcass examinations at five weeks old, weighed, and then slaughtered at the end of the trial. Finally, we measured the weight of the remaining meat and other edible parts.

Blood Biochemical Analysis

The same six birds from every group fasted all night long before being slaughtered for blood collection. Blood samples were gathered in tubes, not including heparin and centrifuged at 5,000 rpm for 15 minutes at 4 degrees Celsius. The resulting serum, the liquid portion remaining after clotting, was stored at -20 degrees Celsius until biochemical analysis. Following the instructions from commercially available kits, we measured the activity of enzymes in the serum, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Additionally, we measured total protein, albumin, urea, and creatinine levels. The activities of oxidative stress markers like malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione (GSH) were determined using the methods explained in an earlier article of ours (Abd El-Hack et al., 2017).

Microbiological Analysis

Six birds from each group were randomly selected, and their ceca were collected. The cecal tissue from each group was pooled and homogenized. Then, 10 g of this homogenate was mixed with 90 milliliters of sterile water peptone and raved for 30 minutes. The liquid portion was collected, representing a 1:10 dilution (10^-1 dilution). Further dilutions were made in tenfold steps up to a dilution (10^-7) (Abd-El-Hack et al., 2021). Specific media were used to count the total bacterial count (TBC), Lactobacilli, coliforms, Escherichia coli, and Salmonella spp. The following methods are described in Abd El-Hack et al. (2021) and Abou-Kassem et al. (2021). Finally, in logarithmic units, the bacterial numbers were transformed (log CFU/g) as described by Sheiha et al. (2020).

Statistical Analysis

The data was examined using a completely randomized design ANOVA test implemented with the GLM procedures in SAS software (SAS, 2001). To compare group means, we employed Tukey's post-hoc test. Unless otherwise mentioned, we considered results statistically significant at a p-value less than 0.05. The following statistical model was used:

Y_ij = μ + T_i + e_ij

Where Y_ij = the perceived value of the treatment in question, μ = it means observed for the treatment in question, T_i= treatment impact and e_ij = error related to an individual observation.

RESULTS AND DISCUSSION

Growth Performance

Live Body Weight and Body Weight Gain

The impact of formic acid (FA) supplementation on broiler chick weight throughout the experiment is shown in Table 2. Chicks' LBW wasn't statistically different between treatment groups at any point except for one week of age. At that age, the various FA levels considerably affected LBW (P < 0.05). Similarly, no significant variations were observed in BWG amongst treatment groups across most periods. However, the varying FA levels significantly impacted BWG during wk 0 to 1 and 4 to 5 (P < 0.05).

Table 2.

Body weight and body weight gain of broilers as affected by dietary supplementation of antibiotic or formic acid levels during the experimental periods.

	Periods
Items	0 wk	1 wk	2 wk	3 wk	4 wk	5 wk
LBW (g/bird)
NC	45.33	199.00ab	499.17	983.22	1662.33	1938.46
PC	45.67	196.17b	474.00	981.83	1679.00	2025.33
FA2	45.80	202.50ab	494.33	951.50	1613.33	1886.72
FA4	44.60	208.83a	487.17	977.57	1671.19	1988.94
FA6	45.87	209.50a	505.17	978.50	1614.83	1978.00
SEM	0.29	1.89	1.72	1.14	1.81	1.96
P value	0.700	0.046	0.282	0.895	0.420	0.113
BWG (g/ bird/ day)	0–1 wk	1–2 wk	2–3 wk	3–4 wk	4–5 wk	0–5 wk
NC	21.95ab	42.88	69.15	97.02	69.03b	59.16
PC	21.50b	39.69	72.55	99.59	86.59ab	61.87
FA2	22.39ab	41.69	65.31	94.55	68.35b	57.53
FA4	23.46a	39.76	70.06	99.09	79.44ab	60.76
FA6	23.38a	42.24	67.62	90.91	90.80a	60.38
SEM	0.27	0.56	0.15	0.67	0.43	0.36
P value	0.045	0.264	0.385	0.508	0.012	0.111

-NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin antibiotic/ kg diet); FA2, FA4 and FA6: basal diet + 2, 4 and 6 cm³ formic acid/ kg diet, respectively. LBW: live body weight; BWG: daily body weight gain.

-Means in the same column within each classification bearing different letters are significantly different.

Our findings disagree with those of Gheisar et al. (2015), who observed the positive influences of OAs on poultry performance, particularly in broiler chickens. They attributed this benefit to the antimicrobial activity of OAs, suggesting it improved the birds' gut microflora and nutrient availability. They also proposed that OAs might enhance broiler BWG by affecting the integrity of the microbial cell membranes. This improvement might also be caused by OAs interfering with the bird's energy metabolism and nutrient transport. For instance, a report by Ragaa et al. (2016) presented that adding formic acid (5 g/kg) to a basal diet significantly increased BWG.

Similarly, Rashid et al. (2020) observed no significant differences in broiler performance between treatment groups at 21-days-old. However, at 42 d, birds fed diets with OAs displayed a gradual rise in BWG and a better FCR. Further supporting this, Nguyen and Kim (2020) studied different levels of a mixture of OAs in chicken feed. Their experiment used a basal corn-soybean meal diet and supplemented versions with varying amounts of the OA and medium-chain fatty acids (MCFAs) blend (0.25, 0.5, 0.75, and 1 g / kg of diet). They found that including the OA blend in the basal diet resulted in a rise (p<0.05) in BWG. Also, Stamilla et al. (2020) investigated how adding a mix of OAs and essential oils (EO) to broiler feed impacts their growing and gut health. Their study showed that this treatment of OAs and EO led to chickens gaining weight faster on average (p < 0.01). This aligns with other research, such as Hernández et al. (2006), who established that including fit for 10,000 ppm of formic acid in broiler feed improved their ability to digest dry matter. However, Hernández et al. (2006) also detected that 10 g / kg FA didn't affect the pH of the small intestine or the microscopic structure of the chickens' intestines.

Feed Intake and Feed Conversion Ratio

Table 3 shows how formic acid supplementation affected the FI and FCR throughout the study. The groups' FI and FCR did not significantly differ from one another at any point except for the 1 to 2 wk of age. During that time, FI and FCR were considerably impacted by the different quantities of formic acid added to the feed.

Table 3.

Feed intake and feed conversion ratio of broilers as affected by dietary supplementation of antibiotic or formic acid levels during the experimental periods.

	Periods
Items	0–1 wk	1–2 wk	2–3 wk	3–4 wk	4–5 wk	0–5 wk
FI (g/ day)
NC	24.13	56.36b	96.48	148.81	148.05	94.77
PC	24.13	63.44a	98.78	154.56	172.15	102.61
FA2	24.13	58.12b	99.14	151.33	136.89	93.92
FA4	24.13	56.56b	96.49	150.94	158.55	97.33
FA6	24.13	58.98b	98.55	155.92	163.53	100.22
SEM	0.13	0.83	1.67	1.88	1.09	1.79
P value	0.990	0.013	0.984	0.958	0.211	0.563
FCR (g feed/ g gain)	0-1 wks.	1-2 wks.	2-3 wks.	3-4 wks.	4-5 wks.	0-5 wks.
NC	1.10	1.32b	1.40	1.54	2.16	1.60
PC	1.13	1.60a	1.36	1.55	2.03	1.66
FA2	1.08	1.39b	1.53	1.60	2.01	1.63
FA4	1.03	1.42b	1.37	1.52	2.02	1.60
FA6	1.03	1.40b	1.46	1.73	1.88	1.67
SEM	0.01	0.03	0.04	0.03	0.09	0.03
P value	0.837	0.018	0.692	0.722	0.341	0.638

-NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin® antibiotic/ kg diet); FA2, FA4 and FA6: basal diet + 2, 4 and 6 Cm³ formic acid/ kg diet, respectively. FI: feed intake; FCR: feed conversion ratio.

-Means in the same column within each classification bearing different letters are significantly different.

Our results concur with the findings reported by, who saw a decrease in broilers' feed intake when using the salt form of these organic acids. However, growth remained similar to control birds, and the FCR improved (Paul et al., 2007). They also observed that a combination of OAs in the broiler diet led to a linear improvement in FCR at seven days old (p ≤ 0.05), but it didn't influence performance afterward the first week. But, Rashid et al. (2020) stated a lower FCR in treatment groups compared to the control group at 42 d. Also, our findings differ from those of Stamilla et al. (2020), who examined the impact of supplementing broiler chicken diets with a blend of OAs and EO on productive performance. They observed that the OAs and EO group exhibited improved BWG (p < 0.01) and FCR between 37 and 47 d compared to the control group.

Similarly, Nguyen and Kim (2020) studied the effects of adding a mixture of OAs and MCFAs to the diet of one-day-old Ross 308 broiler chicks over 7 wk. Their research indicated that including the OA and MCFA blend in the basal diet caused an improvement in FCR. As the amount of OA and MCFA mixture increased in the chickens' diets, their feed intake steadily declined over the first 7 d (p = 0.002). This aligns with a study by Pham et al. (2020), who explored the impact of adding a mixture of organic acids and encapsulated essential oils (BLJ) to the diet on broiler chickens' growth in a model with a combined Necrotic enteritis (NE) infection. They observed that the supplemented diet consistently improved FCR throughout the experiment (P < 0.01).

Also, the authors found that adding BLJ to the feed significantly enhanced the FCR. However, Paul et al. (2007) reported a different outcome – a reduction in FI for broilers when 3 g/kg of Ammonium format (an ammonium salt of formic acid) was included in their feed. Interestingly, this addition also improved LBW and FCR.

Carcass Traits

After the experiment, Table 4 shows how adding formic acid to the chickens' diet affected their carcass characteristics. All the measurements we looked at - the weight of the whole carcass, dressing percentage (meat weight after feathers are removed), breast meat, thigh meat, spleen, and giblets were significantly different (P < 0.05) depending on how much formic acid the chicks were fed. The chicks that ate feed with the highest level of formic acid (6 cm³/kg of food) had the heaviest carcasses and the highest dressing percentage, compared to the chicks that didn't get any formic acid and the chicks that ate food with other amounts of formic acid. Interestingly, the amount of formic acid didn't affect other measurements. For example, the chicks that ate the middle amount of formic acid had the maximum percentage of breast meat compared to the control group and the chicks that ate different amounts of formic acid.

Table 4.

Carcass traits of broilers as affected by dietary supplementation of antibiotic or formic acid levels during the experimental periods.

	As a % of the slaughter weight
Items	Carcass	Giblets	Dressing	Breast	Thigh	Liver	Gizzard	Heart	Spleen
NC	72.32b	4.09a	76.42b	61.43b	38.44a	3.10a	1.67	0.69	0.19a
PC	72.35b	4.11a	76.46b	61.74b	38.26a	3.50a	1.46	0.60	0.12b
FA2	72.78b	4.14a	76.93ab	63.51a	36.38b	3.39a	1.64	0.52	0.15ab
FA4	73.85ab	4.13a	77.99a	63.78a	36.03b	3.23a	1.65	0.59	0.13b
FA6	74.53a	3.53b	78.06a	61.44b	38.15a	2.65b	1.37	0.60	0.13b
SEM	0.29	0.08	0.24	0.35	0.33	0.09	0.07	0.02	0.01
P value	0.024	0.019	0.029	0.027	0.010	0.012	0.590	0.218	0.021

-NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin® antibiotic/ kg diet); FA2, FA4 and FA6: basal diet + 2, 4 and 6 Cm³ formic acid/ kg diet, respectively.

-Means in the same column within each classification bearing different letters are significantly different.

Our results align with Rashid et al. (2020), who studied the impact of organic growth-promoting agents like phytogenic feed additives and OAs on broiler chickens over 42 d. They observed that including these additives, both alone and combined, increases carcass and breast meat output. On the other hand, Nguyen and Kim (2020) looked at how a combination of MCFAs and OAs affected young broiler chickens. Still, they found no significant impact on carcass value.

Blood Biochemical Parameters

Liver and Kidney Functions

Blood chemistry, measured through biochemical parameters, is a window into a bird's health. These parameters reflect the bird's nutritional status, physiological functions, and underlying diseases. They can be a valuable tool to understand how diet, including specific nutrients and additives, affects the bird's health. Table 5 reviews the impact of fatty acid supplements on the blood chemistry of broiler chicks. The levels of urea, creatinine, total protein, globulin, and albumin in the blood serum and the activity of liver enzymes ALT and AST differed significantly between chicks fed the FA compared to those in the control groups (PC and NC).

Table 5.

Blood biochemical indices of broilers as affected by dietary supplementation of antibiotic or formic acid levels at five weeks of age.

Items	Total protein (mg/dL)	Albumin (mg/dL)	Globulin (mg/dL)	A/ G ratio	AST (IU/L)	ALT (IU/L)	Urea (mg/dL)	Creatinin (mg/dL)
NC	4.35a	2.45a	1.90a	1.29a	53.50a	105.00b	12.00a	0.53a
PC	4.15b	2.25b	1.90a	1.19a	52.50a	119.00a	12.50a	0.47b
FA2	3.05d	1.45c	1.60b	0.92b	31.50b	64.50c	8.00b	0.54a
FA4	2.60e	1.20d	1.40c	0.86b	26.00c	46.00e	8.00b	0.34d
FA6	3.40c	1.60c	1.80a	0.89b	19.00d	55.50d	6.50c	0.39c
SEM	0.18	0.13	0.06	0.05	0.77	0.71	0.64	0.02
P value	0.000	0.001	0.000	0.000	0.001	0.001	0.000	0.000

-NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin® antibiotic/ kg diet); FA2, Fa4 and Fa6: basal diet + 2, 4 and 6 Cm³ formic acid/ kg diet, respectively. AST: aspartate transaminase; ALT: alanine transaminase.

-Means in the same column within each classification bearing different letters are significantly different.

In a 42-d study by Rashid et al. (2020), researchers investigated the impact of organic growth-promoting agents, including plant-based feed additives (phytogenic) and OAs, on broiler chickens. They looked at how these supplements affected the chickens' performance, the characteristics of their carcasses, blood parameters, the makeup of their gut bacteria, and how these microbes interacted with the chickens themselves. Interestingly, most biochemical markers they examined displayed no significant variation between the control and treatment groups. However, the one exception was the albumin-to-globulin ratio (A/G ratio), which was more (P < 0.05) in the groups that received the natural growth promoters.

In a study on how formic acid affects broiler growth, Ragaa et al (2016) investigated the influences of adding formic acid (FA) and potassium diformate (KDF) to broiler feed. Their research looked at performance, carcass characteristics, blood chemistry, gut bacteria levels, intestinal tissue structure, and immune function in the birds. They found that both FA and KDF supplementation significantly impacted performance, immune response, and gut health, but there wasn't a major effect on blood chemistry. Separately, Saki et al. (2023) reported that adding natural growth promoters (NGPs), which include organic acids and plant-based feed additives, to chicken diets helped maintain healthy blood cholesterol levels and albumin-to-globulin ratio (A/G ratio). However, more recent research by Negm et al. (2023) showed that the birds' type of feed influenced all blood chemistry markers. Their study found that birds fed a specific diet (LAC2) had their blood's highest total protein and albumin levels. They also observed that increasing amounts of LAC to broiler feed lowered liver enzyme activity and blood urea concentration.

Antioxidant Parameters

This study investigated how adding formic acid (FA) to broiler chickens' diets affected their antioxidant levels. The activities of enzymes like glutathione peroxidase (GPx), glutathione S-transferase (GST), and the concentration of glutathione (GSH) were all measured. Malondialdehyde (MDA), an oxidative stress marker, was also assessed. Tables 6 show the detailed results. Interestingly, all these antioxidant parameters were considerably influenced (p < 0.05) by the amount of formic acid supplementation compared to the control and negative control groups. There was also a slight decrease in MDA levels as the formic acid supplementation increased.

Table 6.

Blood antioxidant parameters of broilers as affected by dietary supplementation of antibiotic or formic acid levels at five weeks of age.

Items	GPx (nmol/L)	GST (nmol/L)	GSH (nmol/L)	MDA (nmol/L)
NC	152.50d	4.60c	62.00b	6.05a
PC	169.38b	5.65b	73.25a	5.50a
FA2	160.00c	6.40a	74.00a	3.80bc
FA4	157.00cd	4.09d	62.50b	4.29b
FA6	179.00a	5.85b	71.50a	3.42c
SEM	2.62	0.23	1.50	0.278
P value	0.000	0.001	0.001	0.000

-NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin® antibiotic/ kg diet); FA2, FA4 and FA6: basal diet + 2, 4 and 6 Cm³ formic acid/ kg diet, respectively. GPx: glutathione peroxidase; GST: glutathione-S-transferase; GSH: reduced glutathione MDA: malondialdehyde

-Means in the same column within each classification bearing different letters are significantly different.

Likewise, Arif et al. (2016, 2019) discovered that including 2.25 grams per kilogram of humic acid in broiler diets reduced liver enzyme activity. Some OAs, such as FA, have been shown to develop the antioxidant status of chickens. This might be due to their ability to influence gut microbiota, decreasing the growth of injurious bacteria and promoting beneficial ones (Elnaggar and El-kelawy, 2024). Also, studies have shown elevated levels of antioxidant enzymes in chicken-fed diets containing OAs, such as catalase (CAT) and superoxide dismutase (SOD). These enzymes help neutralize harmful free radicals in the body (Apalowo et al., 2024).

Microbial Analysis in Broiler Cecum

All bacterial populations, except Salmonella were considerably impacted (P < 0.01) by the treatments (Table 7). The FA2 group had the maximum TBC. In contrast, the FA6 group had the lowest levels of pathogenic bacteria (E. coli and Coliform).

Table 7.

Cecal microbial count of broilers as affected by dietary supplementation of antibiotic or formic acid levels at five weeks of age.

	Microbial count (Log10 CFU/ g)
Items	TBC	Lactobacilli	Coliform	Escherichia coli	Salmonella
NC	8.30c	7.32b	6.44a	5.41a	4.42
PC	8.27c	7.21c	6.24c	5.21b	4.14
FA2	8.43a	7.40a	6.37b	4.35c	3.85
FA4	8.37b	7.38a	6.37b	4.37c	4.41
FA6	8.06d	7.06d	6.05d	4.12d	4.25
SEM	0.03	0.03	0.04	0.14	0.09
P value	0.001	0.000	0.002	0.000	0.201

- NC: negative control (basal diet); PC: positive control (basal diet + 0.5 g Colistin® antibiotic/ kg diet); FA2, FA4 and FA6: basal diet + 2, 4 and 6 Cm³ formic acid/ kg diet, respectively. TBC: total bacterial count.

-Means in the same column within each classification bearing different letters are significantly different.

Bird feed has been successfully supplemented with organic acids for years to combat pathogens, particularly Salmonella bacteria and fungal mycotoxins. But recent research suggests organic acids do even more – they can directly reduce harmful bacteria within the bird's intestines (Dittoe et al., 2018). Our findings align to some degree with Attia et al. (2018), which showed that OAs can adjust the pH of the gut, hindering the growth of bad bacteria and promoting a healthy balance of microbes in the digestive system (GIT). Research suggests that organic acids can be a weapon against harmful bacteria in chickens. Studies by Saki et al. (2012) have shown that organic acids help reduce Salmonella spp. and Enterobacteriaceae bacteria in broiler chickens. Ricke (2003) found that formic and propionic acid had a bactericidal effect against Salmonella spp., Coliforms, and E. coli in the bird's digestive tract (GIT).

Additionally, Nair and Kollanoor (2019) suggest that including OAs in a chicken's diet can help prevent the vertical transmission of Salmonella. Similarly, Regassa and Nyachoti (2018) reported that adding butyric acid to a bird's diet can reduce Salmonella Enteritidis infection within the GIT. Also, adding up to 30 g/kg of citric and fumaric acid to broiler feed can significantly reduce the total bacteria and Enterobacteriaceae in their cecum. However, the study by Attia et al. (2018) recommends a more cost-effective level of 15 g/kg for practical use.

Studies have shown that OAs can be a beneficial alternative to antibiotics for promoting gut health in animals. Palamidi and Mountzouris (2018) found that adding OAs improved the balance of microbes in the cecum and boosted the activity of helpful digestive enzymes produced by bacteria in the ileum. Interestingly, their research also showed that treatment with OAs significantly increased beneficial bacterial groups like Clostridium leptum and Clostridium coccoides in the cecum, compared to treatment with avilamycin. The positive effects of OAs likely stem from their capability to reduce the pH of the digestive tract. Certain OAs, like lactic, fumaric, and citric acids, create a more acidic environment that discourages the growth of harmful bacteria sensitive to pH changes in the stomach (Dittoe et al., 2018).

Additionally, other OAs such as formic, acetic, butyric, propionic, and sorbic acids can directly target the cell walls of Gram-negative bacteria, further reducing their numbers. Research by Mohamed Negm et al. (2023) supports this concept, demonstrating that supplementing with lactic acid (LAC) had an even greater impact on reducing harmful bacteria (E. coli and Salmonella) compared to antibiotics. This suggests that LAC may be particularly effective for promoting gut health.

CONCLUSIONS

Our findings show that adding formic acid to the diet didn't significantly impact most growth factors. However, it may improve blood parameters, oxidative conditions, and gut bacteria levels. Constructed on our outcomes, the recommended level for the best health outcomes in the final product is the medium level (4.0 Cm³ FA/kg diet).

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.