Isolated and combined organic acids in diets of broiler chickens challenged with Eimeria acervulina

[Ácidos orgânicos isolados ou associados em dietas de frangos de corte desafiados com Eimeria acervulina]

ABSTRACT

This study aimed to evaluate the effect of isolated or combined citric and benzoic acids added to the diets of broiler chickens on performance, allometry of the digestive system organs, intestinal pH and quantity of microorganisms in the jejunum. A total of 840 male Cobb broiler chicks were utilized, distributed in a complete random design in 2 × 2 factorial scheme, supplemented or not with citric acid, and sodium benzoate, with seven replications. At 14 days old, 1mL of a solution containing 1 × 10⁵ sporulated oocysts of Eimeria acervulina per bird was inoculated orally. There was no effect of the acids on the broiler’s performance in the 1 to 21-day period. In the total period (1 to 42 days), the broilers fed with a blend of citric and benzoic acid presented greater feed intake, without increment in weight gain. The data of allometry of the digestive system organs and the jejunal pH values were not influenced by the treatments. The citric acid increased the bacterial quantity of gram-positive coccus and total anaerobes in the jejunum. Under the conditions of the present study, we conclude that the citric and benzoic acids, isolated or combined, do not benefit the nutrition of broilers challenged with E. acervulina.

Keywords:
acidifying; additive; gastrointestinal tract; microbiota; performance; poultry

RESUMO

O objetivo deste trabalho foi avaliar os efeitos da inclusão isolada ou associada dos ácidos cítrico e benzoico na alimentação de frangos de corte sobre o desempenho, a alometria de órgãos do sistema digestório, o pH intestinal e a quantidade de microrganismos no jejuno. Foram utilizados 840 pintos de corte, machos, da linhagem Cobb, distribuídos num delineamento inteiramente ao acaso, em esquema fatorial 2 × 2, com suplementação ou não de ácido cítrico e suplementação ou não de benzoato de sódio, com sete repetições. Aos 14 dias de idade, foi inoculado, via oral, 1mL de solução contendo 1 × 10⁵ oocistos esporulados de Eimeria acervulina por ave. Não houve efeito dos ácidos sobre o desempenho dos frangos no período de um a 21 dias. No período total (um a 42 dias), os frangos alimentados com a mistura de ácidos cítrico e benzoico apresentaram maior consumo de ração, sem incremento no ganho de peso. Os dados de alometria dos órgãos do sistema digestório e os valores de pH do jejuno não foram influenciados pelos tratamentos. O ácido cítrico aumentou a quantidade de bactérias do gênero cocos Gram positivos e anaeróbios totais no jejuno. Nas condições do presente estudo, conclui-se que os ácidos cítrico e benzoico, isolados ou associados, não beneficiam a nutrição de frangos de corte desafiados com E. acervulina.

Palavras-chave:
acidificante; aditivo; ave; desempenho; microbiota; trato gastrintestinal

INTRODUCTION

One of the main current challenges in poultry production is the search for balance between the intestinal microbiota and the host. Within this context, organic acids have been studied to model the intestinal microbial population, assisting the good functioning of the organism and the performance of broiler chickens (Barbosa et al., 2005). The organic acids inhibit the development of fungi in raw materials and feeds, decreasing the proliferation of enterobacteria (Salmonella and Escherichia coli) in the intestine, reflecting in a greater nutritional utilization of rations (Gonzales and Sartori, 2001).

The inhibition of the microbial growth by organic acids is explained by the ability of these acids to go through the membrane, dissociating in the cellular interior, acidifying the cytoplasm (Van Immerseel et al.; 2006). The excessive export of protons by the bacteria to control the intracellular pH demands intake of adenosine triphosphate (ATP), which results in the depression of cellular energy (Ricke 2003), retarding its growth or even causing the microorganism to die. The benefits of acids on the control of pathogenic microorganism were reported by Flores et al. (2012) who observed a decrease in the presence of Salmonella in broiler chickens treated with organic acids.

Many studies investigate the effect of organic acids on broiler chickens’ performance (Waldroup et al.; 1995; Rezende et al.; 2008; Chowdhury et al.; 2009; Adil et al.; 2010; Barbieri et al.; 2015). However, the published results are controversial, which does not allow a safe recommendation regarding the utilization and concentration of these products. The lack of consistency in the results of organic acids could have resulted from a lack of control in the intervening variables such as pH in the digestive tract, buffer capacity of diet ingredients, hygiene conditions in the production environment, heterogeneity of the intestinal microbiota, besides the inherent resistance of the microorganisms. Another important point is to investigate the effects of organic aids on Eimeria, which is an intestinal pathogen that causes the most damages in poultry production (Williams, 1999).

Therefore, this research study aimed to evaluate the supplementation of citric and benzoic acids, isolated or combined, in broiler chickens challenged with Eimeria acervulina on the performance, allometry of the digestive system organs, length of small and large intestines, pH of the intestinal content, and on the development of the microbiota found in the gastrointestinal tract (GIT).

MATERIAL AND METHODS

The study was carried out at the São Paulo State University (UNESP), College of Agricultural and Technological Sciences, Dracena, with the following geographical coordinates: latitude 21º 28’ 57” south, longitude 51º 31’ 58’’ west and average altitude of 421 meters. The climate in the region is subtropical (warm and dry winter is followed by very hot summers) with average annual temperature of 23.6ºC according to the technical manual developed by Tremocoldi, Brunini (2008). The experiment was carried out according to the principles and regulations of the Ethics Committee for the Use of Animals-CEUA, São Paulo State University (UNESP), College of Agricultural and Technological Sciences, Dracena (Registration No. 38/2012).

The broiler chicks were housed in floor pens and raised on the floor with new wood shaving litter. The management practices followed the technical orientation of the Cobb manual. The initial heating of the chicks was done through 250-watt infrared lamps. Along the experiment, the control of the temperature and ventilation was manually done by managing the lateral curtains of the experimental rearing house and utilizing fans. The lighting was constant with 60-watt incandescent light bulbs.

A total of 840 1-day-old male Cobb broiler chicks were utilized from a commercial incubator, previously vaccinated against infectious bursal disease, Marek’s disease, and Bouba’s disease. The chicks were distributed in a completely random design in a 2x2 factorial scheme, with or without supplementation of citric acid and sodium benzoate with seven replications per treatment, resulting in 28 floor pens of 2.5m², with 30 birds per floor pen (experimental unit), at initial density of 12 birds/m². The treatments were: basal feed (control treatment) - without addition of additives, basal feed + addition of 0.500% of citric acid, basal feed + addition of 0.242% of sodium benzoate, and basal feed + addition of a blend of citric acid and sodium benzoate (total of 0.742% of acids). The addition of acids started from the substitution of inert material (kaolin). Both citric and sodium benzoate acids were in powder form and were incorporated to the feed at the time of processing.

The feeding program was divided into four phases: pre-starter, 1 to 7 days; starter, 8 to 21 days; grower, 22 to 33 days; and finisher, 34 to 42 days (Table 1). The diets were formulated with corn and soybean meal, according to the recommendations by Rostagno et al. (2011), without the addition of any type of antibiotic or anticoccidial. Water and feed were supplied ad libitum. Initial drinkers and feeders were utilized and substituted for bell drinkers at three days old, and for tubular feeders at 14 days old. The total period of poultry production was from 1 to 42 days old.

Table 1
Composition and calculated values of the experimental diets

Ingredients, %	Diets¹
	Pre-starter	Starter	Grower	Finisher
Corn	53.94	58.00	60.58	65.40
Soybean meal	38.37	34.97	31.78	27.53
Soybean oil	2.58	2.57	3.57	3.36
Choline chloride 60	0.072	0.064	0.058	0.043
Salt2	0.508/0.409	0.482/0.384	0.457/0.359	0.444/0.346
Dicalcium-phosphate	1.900	1.532	1.341	1.076
Limestone	0.918	0.908	0.820	0.766
L-lysine	0.284	0.212	0.187	0.229
DL-methionine	0.357	0.285	0.254	0.238
L-threonine	0.106	0.058	0.039	0.048
L-valine	0.075	0.024	0.015	0.030
Mineral premix3	0.050	0.050	0.050	0.050
Vitamin premix4, 5, 6 and 7	0.100	0.100	0.100	0.050
Kaolin8	0.742	0.742	0.742	0.742
Sum	100	100	100	100
	Calculated values
AMEn, kcal/kg	2,950	3,000	3,100	3,150
CP, %	22.20	22.80	19.50	18.00
Lysine9, %	1.310	1.174	1.078	1.010
Methionine9, %	0.646	0.562	0.518	0.486
Methionine+cystine9, %	0.944	0.846	0.787	0.737
Threonine9, %	0.852	0.763	0.701	0.656
Valine9, %	1.010	0.904	0.841	0.788
Calcium, %	0.920	0.819	0.732	0.638
Phosphorus9, %	0.395	0.343	0.313	0.273
Sodium, %	0.220	0.210	0.200	0.195
Choline, mg/kg	375.0	330.0	300.0	225.0
Linoleic acid, %	2.73	2.77	3.32	3.26

• ¹
  Pre-starter, 1 to 7 d-old; starter, 8 to 21 d-old; Grower, 22 to 33 d-old; Finisher, 34 to 42 d-old. ²There was a reduction in the NaCl content of diets with inclusion of sodium benzoate to compensate the sodium present in the additive.³Mineral premix provided per kg of feed: Cu, 8; Fe, 50; Mn, 70; Zn, 50; I, 1.2; and Se, 0.2mg. ⁴Vitamin premix provided per kg of feed in pre-starter diet (1-7 d): vitamin A, 8,000.00IU; vitamin D3, 2,400.00IU; vitamin E, 12.00IU; vitamin K3, 2.00mg; vitamin B1, 2.40mg; vitamin B2, 6.00mg; vitamin B6, 4.00mg; vitamin B12, 14.00mcg; folic acid, 1.00mg; pantothenic acid, 15.00mg; niacin, 40mg. ⁵Vitamin premix provided per kg of feed in starter diet (8-21d): vitamin A, 7,000.00IU; vitamin D3, 2,200.00IU; vitamin E, 11.00IU; vitamin K3, 1.60mg; vitamin B1, 2.00mg; vitamin B2, 5.00mg; vitamin B6, 3.00mg; vitamin B12, 12.00mcg; folic acid, 0.80mg; pantothenic acid, 13.00mg; niacin, 35mg.⁶Vitamin premix provided per kg of feed in grower diet (22-33d): vitamin A, 6,000.00IU; vitamin D3, 2,000.00IU; vitamin E, 10.00IU; vitamin K3, 1.60mg; vitamin B1, 1.40mg; vitamin B2, 4.00mg; vitamin B6, 2.00mg; vitamin B12, 10.00mcg; folic acid, 0.60mg; pantothenic acid, 11.00mg; niacin, 30mg.⁷Vitamin premix provided per kg of feed in finisher diet (34-42d): vitamin A, 5,000.00IU; vitamin D3, 1,000.00IU; vitamin E, 8.00IU; vitamin K3, 1.60mg; vitamin B2, 2.00mg; vitamin B12, 5.00mcg; pantothenic acid, 9.00mg; niacin, 20mg.⁸Treatments were obtained by replacing kaolin with additives: Diets with citric acid, 0.500%; diets with sodium benzoate: 0.242%; diets with citric acid + sodium benzoate: 0.742%.⁹Digestible values.

In order to provide microbiological challenge to the birds, at 14 days old, 1mL of solution containing 1 × 10⁵ of sporulated oocysts of E. acervulina was inoculated per bird. The broilers were manually contained and orally inoculated using an automatic pipette. The oocysts were acquired from the Laboratory of Molecular Biology of the School of Veterinary Medicine of USP - campus of São Paulo.

The performance data were obtained for the accumulated periods of 1 to 21 and 1 to 42 days old. They were: body weight (weight of the birds for each floor pen in the housing, at 21 and at 42 days old), weight gain (difference between the weight at the end of each period and the initial weight in the housing), feed intake (difference between the total provided feed and the leftovers collected at the end of each period, based on the average number of chickens), feed to gain ratio (ratio between the total consumed feed and the weight gain, corrected by the weight of the dead chickens), viability (100 - mortality that was recorded daily and expressed in percentage, by the relation between the initial number of birds and the number of dead birds in the period), and production factor (average daily weight gain × viability) / feed to gain ratio) / 10).

At 21 and 42 days old, seven birds per treatment were withdrawn (one per replication) and slaughtered by cervical dislocation followed by bleeding to determine the relative weight of the organs, the length of the small and large intestines, and the pH of the intestinal content in order to understand the mechanisms that affect the digestion of nutrients in the evaluated diets. The following organs were collected and weighed: spleen, pancreas, gizzard, proventriculus, liver, small intestine and large intestine. The relative weight of each organ was determined in relation to the birds’ weight. The measurement of the small intestine was done where the duodenum emerges from the gizzard up to the beginning of the cecum. The measurement of the large intestine was calculated by the colon and rectum length added to the result of the caecum length.

To determine pH, 1g of the intestinal content from the initial portion of the jejunum was collected and mixed to 30mL of distilled water in plastic flasks (universal collector). The flasks were agitated and left to rest for a minute and next the reading was done using a portable pH meter (Coon et al.; 1990).

These microbiological analyses were done in the Laboratory of Animal Hygiene of São Paulo University - USP - campus of Pirassununga. At 42 days, a second bird was sacrificed to collect samples from the intestinal content to perform the microbiological analysis in the GIT. The samples were collected from the birds’ jejunum and kept under refrigeration and they were posteriorly analyzed regarding the presence of total enterobacteria, gram-positive cocci and total anaerobes, according to the method described by Danicke et al. (1999).

Data analysis was done by Statistical Analysis System software (SAS/STAT, 2008). Firstly, the analyses of residue normality and variance homogeneity were done. Following the prerequisites, the data were submitted to analysis of variance (ANOVA) with probability at 5% of significance. When needed, the differences between the treatments were studied by comparing the averages of the minimum squares calculated by LSMEANS command with Tukey’s test.

RESULTS AND DISCUSSION

The initial average weight of the chicks was 41.90g. There was no effect of the treatments on the performance of broiler chickens in the period of 1 to 21 days old (Table 2).

The antimicrobial efficiency of the acid depends on its dissociation constant (pKa). This value represents the pH point of the means in which there is balance between the dissociated and non-dissociated form of the acid. According to Partanen’s adapted table (2002), the citric and benzoic acids are considered acids with average pKa with values of 3.13/4.76/6.40 for citric acid and 4.19 for the benzoic acid. For example, in a medium with pH 4.19, the benzoic acid will be half in the dissociated form and half in the non-dissociated one. Therefore, the greater the pKa of an acid is, the more efficient it will be.

Table 2
Performance of broilers at 21-d-old fed diets with or without organic acids

Variables1	Citric Acid	Benzoic Acid		Means	Probability			CV2
		-	+		Citric	Benzoic	Interaction
BW gain	-	871	861	866	0.0511	0.6167	0.1363	2.90
	+	838	857	847
	Means	854	859
FI	-	1,032	1,033	1,032	0.2254	0.6963	0.7381	2.67
	+	1,015	1,023	1,019
	Means	1,024	1,028
F:G	-	1.188	1.207	1.197	0.0561	0.6874	0.1510	2.28
	+	1.222	1.212	1.217
	Means	1.205	1.209
L	-	98.59	98.90	98.74	0.6121	0.2039	0.1017	2.16
	+	99.53	97.14	98.34
	Means	99.06	98.02

• ¹
  BW, body weight (g); FI, feed intake (g); F:G feed:gain (g:g); L, livability (%). ²CV, coefficient of variation (%).

However, despite the characteristics that the citric and benzoic acids present, it was not possible to observe an increment in the initial performance. The absence of interaction also indicated that there was no potentiation with the combination of both acids. In the literature there is evidence that the organic acids included in the isolated form do not produce an effect on the performance, only the blend of several acids improved the feed to gain ratio (Polycarpo et al.; 2017).

It is possible that the action of several acids together present synergy among themselves, although in this study this was not observed. Thus, it is suggested that new combinations be evaluated, testing blends that contain more than two acids.

The results found here corroborate previous studies that did not observe improvement in broilers’ performance with acid blends. Salazar et al. (2008) evaluated the isolated and combined use of butyric and lactic acids and did not observe increment of the broilers’ performance. Vale et al. (2004) evaluated doses of the blend of formic and propionic acids and observed a decrease in the body weight (worsening of the performance), probably due to the feed intake reduction with high doses of acids.

Regarding the dose of organic acids, it is an important point since high doses damage the birds’ intake and consequently their performance (Leeson et al.; 2005; Józefiak et al.; 2007; Islam et al.; 2008; Khosravinia et al.; 2015; Khan and Iqbal, 2016).

In studies carried out with isolated acids, it was also verified that acids do not affect performance. Campos et al. (2004) did not observe the benefits of the increasing doses of fumaric acid in low energy diets for broilers. Leeson et al. (2005) did not verify an improvement in the performance with increasing doses of butyric acid. The performance results of the period of 1 to 42 days are shown in Table 3.

There was interaction of the citric acid with the benzoic acid on the feed intake (FI) (Figure 1). In the interaction study, it is observed that the association of both acids increased the feed intake regarding the isolated use. However, despite the greater intake, the weight gain and feed to gain ratio were not altered by the treatments.

The increase of feed intake can be interpreted as something positive when it is followed by the increase in weight gain, but when it does not change, the increase in the feed intake is not interesting anymore because the cost with feed increases and the production does not alter.

Table 3
Performance of broilers at 42-d-old fed diets with or without organic acids

Variables1	Citric Acid	Benzoic Acid		Means	Probability			CV2
		-	+		Citric	Benzoic	Interaction
BW gain	-	2,648	2,686	2,667	0.5371	0.4206	0.9370	4.07
	+	2,625	2,656	2,641
	Means	2,637	2,671
FI	-	3,800Aa3	3,811Ba	3,806	0.2296	0.0033	0.0101	2.09
	+	3,763Ab	3,906Aa	3,835
	Means	3,781	3,859
F:G	-	1.627	1.628	1.628	0.4792	0.2768	0.2892	3.98
	+	1.618	1.673	1.645
	Means	1.623	1.650
L	-	80.49	77.22	78.85	0.2280	0.1460	0.8200	6.07
	+	82.39	79.98	81.18
	Means	81.44	78.60
PEI	-	312.3	296.9	304.6	0.9993	0.1648	0.6706	13.18
	+	318.9	290.2	304.6
	Means	315.6	293.6

• ¹
  BW, body weight (g); FI, feed intake (g); F:G feed:gain (g:g); L, livability (%); PEI, index of production efficiency (daily weight gain*L)/F:G ratio)/10). ²CV, coefficient of variation (%). ³Different letters, lowercase on the lines and capital on the columns, differ by Tukey test (P<0.05).

Figure 1
Interaction effects of citric and benzoic acid for feed intake at 42 d-old.

Garrido et al. (2004) worked with a blend of two acids (formic + propionic), and in the grower phase observed an increase in feed intake without change in the weight gain. Ribeiro et al. (2008) evaluated a blend of lactic, fumaric and citric acids and did not observe an improvement in the performance. The absence of effect of organic acids can be a result of a series of factors, among them the microbial challenge is a fundamental point to be considered.

Because the benefits of organic acids are mainly related with its antimicrobial effect, it is necessary that there be a microbiological challenge in the experiment for an appropriate evaluation. Since 1950, when the evaluations with antibiotics as growth promoters started, the need for a microbial challenge was evident in Lillie et al. (1953) and Coates et al. (1963), because there is no meaning in evaluating an antimicrobial in an environment without or with little microorganism challenge. In this study, the broilers were challenged with E. acervulina, which causes coccidiosis that is one of the diseases that most causes losses to poultry production. However, facing the results observed in this study, it can be inferred that the absence of increment in the performance by organic acids can be a result of the low bactericidal activity against this pathogen. Eimeria is a protozoa that parasites the intestinal region and it is possible that the evaluated acids reached this region partially or even totally dissociated, which can explain the absence of results.

The data of viability and index of production efficiency were not influenced by the treatments. The low average values of these two variables related to performance occurred due to the birds’ high mortality. This occurred because of the high temperatures recorded in the season of the year in which the experiment was developed (summer), which generated caloric stress and, consequently, an increase in the birds’ mortality.

The allometry results of GIT organs are shown in Table 4 and 5. At 21 as well as at 42 days old, the addition of organic acids do not change the relative weight of organs and the length of intestines. Although organic acids have several additional effects that go beyond those of antibiotics, such as reduction in digesta pH, increased pancreatic secretion, and trophic effects on the gastrointestinal mucosa (Dibner and Buttin, 2002), in this study, alterations on the weight and length of broilers’ intestine were not observed.

On the other hand, Viola et al. (2008) found greater relative weight and length of the small intestine in diets containing blends of organic acids. In rats, it was demonstrated that the presence of short-chain acids stimulate the release of mucus in the colon (Shimotoyodome et al.; 2000). The release of alkaline mucus in the intestine seems to be associated to damages that acids cause in the mucosa (Vattay et al.; 1988). The effect of acids, therefore, can attack the intestinal mucosa and trigger defense reactions, which alter the structure and consequently the intestine’ size, although the results observed here do not make evident the effects on the weight and length of the intestines.

Table 4
Organ relative weight and small and large intestine length of broilers at 21-d-old fed diets with or without organic acids

Variables1	Citric Acid	Benzoic Acid		Means	Probability			CV2
		-	+		Citric	Benzoic	Interaction
Spleen	-	0.105	0.112	0.108	0.5321	0.2319	0.7801	25.79
	+	0.098	0.109	0.104
	Means	0.102	0.110
Pancreas	-	0.415	0.360	0.387	0.3530	0.3428	0.2093	24.81
	+	0.360	0.368	0.364
	Means	0.387	0.364
Gizzard	-	2.21	2.35	2.28	0.8349	0.7748	0.0820	11.15
	+	2.32	2.21	2.27
	Means	2.27	2.29
Liver	-	2.71	2.93	2.82	0.5421	0.4512	0.0829	11.09
	+	2.91	2.83	2.87
	Means	2.81	2.88
Proventriculus	-	0.575	0.564	0.570	0.0882	0.6170	0.8681	22.06
	+	0.525	0.504	0.514
	Means	0.550	0.534
SI (%)	-	7.10	7.78	7.44	0.0767	0.4582	0.0502	12.27
	+	8.04	7.73	7.89
	Means	7.57	7.75
LI (%)	-	1.64	1.81	1.73	0.9141	0.7344	0.0942	25.87
	+	1.87	1.62	1.74
	Means	1.76	1.72
SI (cm)	-	151.50	159.21	155.36	0.7390	0.1912	0.6916	10.74
	+	151.79	155.92	153.86
	Means	151.64	157.57
LI (cm)	-	39.14	42.93	41.04	0.9871	0.4096	0.3741	19.69
	+	41.14	41.00	41.07
	Means	40.14	41.96

• ¹
  SI, small intestine; LI, large intestine. ²CV, coefficient of variation (%).

Table 5
Organ relative weight and small and large intestine length of broilers at 42-d-old fed diets with or without organic acids

Variables1	Citric Acid	Benzoic Acid		Means	Probability			CV2
		-	+		Citric	Benzoic	Interaction
Spleen	-	0.088	0.080	0.084	0.3375	0.5405	0.5177	25.96
	+	0.090	0.090	0.090
	Means	0.089	0.085
Pancreas	-	0.200	0.192	0.196	0.7742	0.4708	0.9186	18.90
	+	0.198	0.186	0.192
	Means	0.199	0.189
Gizzard	-	1.29	1.27	1.28	0.7408	0.8919	0.7520	12.30
	+	1.29	1.30	1.29
	Means	1.29	1.28
Liver	-	1.81	1.74	1.77	0.6127	0.9789	0.2361	12.14
	+	1.71	1.78	1.74
	Means	1.76	1.76
Proventriculus	-	0.26	0.26	0.26	0.5244	0.7035	0.8221	10.98
	+	0.25	0.26	0.26
	Means	0.26	0.26
SI (%)	-	4.58	4.04	4.30	0.7561	0.6810	0.2050	18.71
	+	4.27	4.54	4.41
	Means	4.42	4.29
LI (%)	-	0.88	0.79	0.83	0.4908	0.3798	0.2957	22.11
	+	0.87	0.87	0.87
	Means	0.87	0.83
SI (cm)	-	189.14	192.00	190.57	0.3087	0.5828	0.8925	8.99
	+	195.29	200.00	197.64
	Means	192.21	196.00
LI (cm)	-	45.29	43.50	44.39	0.7791	0.7791	0.3221	11.60
	+	43.50	44.50	44.00
	Means	44.39	44.00

• ¹
  SI, small intestine; LI, large intestine. ²CV, coefficient of variation (%).

In the analyses of pH at 21 and 42 days, the effect of organic acids was not observed (Table 6). The measurement of intestinal pH was done in the jejunum because most of the nutrients are absorbed in this segment.

The absence of effect of acids on the pH confirms the hypothesis that acids be dissociated in the proximal parts of GIT, not influencing the intestine. El-Ghany et al. (2016) found a reduction in the pH values of intestines with the utilization of organic acids, and as a result, bacterial growth was disturbed.

The greater amount of combined acids may have favored the selection of acid pH microorganisms in the proximal portions, reflecting in the microbial population and lower pH values in most distal regions of the intestine.

There was no interaction of acids on the total counting of the bacteria in the samples from the jejunum to 42 days (Table 7). The citric acid, regardless of being associated to benzoic acid, favored the increase of gram-positive cocci and total anaerobes in the jejunum. The benzoic acid did not change the results of the microbiological analyses. There was not growth and formation of total enterobacterial colonies, but it is important to emphasize that the lack of enterobacterial colonies does not mean that bacteria of this kind were not found in the birds’ intestine. These results indicated that the amount of total enterobacteria was low, not reaching the limit for analysis detection.

Table 6
Intestinal pH of broilers at 21 and 42-d-old fed diets with or without organic acids

Variables	Citric Acid	Benzoic Acid		Means	Probability			CV1
		-	+		Citric	Benzoic	Interaction
pH 21dias	-	6.39	6.51	6.45	0.2988	0.1872	0.3767	2.22
	+	6.38	6.41	6.39
	Means	6.39	6.46
pH 42dias	-	6.93	7.00	6.96	0.9893	0.8296	0.0974	1.30
	+	6.99	6.94	6.96
	Means	6.96	6.97

• ¹
  CV, coefficient of variation (%).

Table 7
Total count of bacterial in jejunum from broilers at 42-d-old fed diets with or without organic acids

Bacteria1 (CFU g/ jejunum)	Citric Acid	Benzoic Acid		Means	Probability			CV2
		-	+		Citric	Benzoic	Interaction
GPC	-	3.21×102	3.67×102	3.42×102	0.0019	0.064 1	0.1036	68.05
	+	6.50×102	13.07×102	10.04×102
	Means	4.73×102	8.73×102
TA	-	27.14×102	44.71×102	35.93×102	0.0279	0.3993	0.1422	43.16
	+	55.71×102	50.83×102	53.46×102
	Means	41.43×102	47.54×102

• ¹
  CFU/g, colony forming units per g of sample. Data submitted to log transformation; GPC, gram-positive cocci; TA, total anaerobes. ²CV, coefficient of variation (%).

The results on the intestinal microbiota contradict the ones found by Polycarpo et al. (2016), who did not observe alterations of gram-positive cocci and total anaerobes with addition of organic acids to the diets. Aydin et al. (2010) observed that the addition of 3% of citric acid to the diet did not alter the intestinal microbiota, and also argued that this result is due to the action mode of citric acid, which mainly occurs in the upper part of GIT. The divergence between the results of this study and the ones found in the literature, suggests that more research be done to extend the reflections of citric acid supplementation in broilers’ intestinal microbiota.

CONCLUSION

Citric and benzoic acids, isolated or combined, do not provide improvement in the performance of broiler chickens. The feed intake increases with the inclusion of the blend of citric and benzoic acids, without increment in the weight gain. Citric and benzoic acids do not modify the allometry of the digestive system organs as well as the intestinal pH. Only the citric acid favors greater concentrations of gram-positive bacteria and total anaerobes in chicken’s jejunum. With this study, it was concluded that citric and benzoic acids do not benefit the nutrition of broilers challenged with E. acervulina.

ACKNOWLEDGEMENTS

The authors thank São Paulo Research Foundation (FAPESP, Brazil) for their financial support (Proc 2012/16193-0). We also thank Nutriacid/Btech for donating the organic acids.

REFERENCES

• ADIL, S.; BANDAY, T.; BHAT, G.A.; MIR, M.S.; REHMAN, M. Effect of dietary supplementation of organic acids on performance, intestinal histomorphology, and serum biochemistry of broiler chicken. Vet. Med. Int., v.2010, p.1-7, 2010.
• AYDIN, A.; PEKEL, A.Y.; ISSA, G.; DEMIREL, G.; PATTERSON, P.H. Effects of dietary copper, citric acid, and microbial phytase on digesta pH and ileal and carcass microbiota of broiler chickens fed a low available phosphorus diet. J. Appl. Poult. Res., v.19, p.422-431, 2010.
• BARBIERI, A.; POLYCARPO, G. DO V.; CARDOSO, R.G.A. et al. Effect of probiotic and organic acids in an attempt to replace the antibiotics in diets of broiler chickens challenged with eimeria spp. Int. J. Poult. Sci., v.14, p.606-614, 2015.
• BARBOSA, N.A.A.; SAKOMURA, N.K.; SANTOS, F.R. et al. Efeito da associação de ácidos orgânicos sobre o desempenho de frangos de corte. Rev. Bras. Ciênc. Avíc., v.7, p.123, 2005.
• CAMPOS, M.P.A.; RABELLO, C.B.V.; SAKOMURA, N.K. et al. Utilização do ácido fumárico em dietas de frangos de corte com baixa energia metabolizável. Acta Sci. Anim. Sci., v.26, p.35-39, 2004.
• CHOWDHURY, R.; ISLAM, K.M.S.; KHAN, M.J. et al. Effect of citric acid, avilamycin, and their combination on the performance, tibia ash, and immune status of broilers. Poult. Sci., v.88, p.1616-1622, 2009.
• COATES, M.E.; FULLER, R.; HARRISON, G.F.; LEV, M.; SUFFOLK, S.F. A comparision of the growth of chicks in the Gustafsson germ-free apparatus and in a conventional environment, with and without dietary supplements of penicillin. Br. J. Nutr., v.17, p.141, 1963.
• COON, C.N.; LESKE, K.L.; AKAVANICHAN, O.; CHENG, T.K. Effect of oligosaccharide-free soybean meal on true metabolizable energy and fiber digestion in adult roosters. Poult. Sci., v.69, p.787-793, 1990.
• DANICKE, S.; VAHJEN, W.; SIMON, O.; JEROCH, H. Effects of dietary fat type and xylanase supplementation to rye-based broiler diets on selected bacterial groups adhering to the intestinal epithelium, on transit time of feed, and on nutrient digestibility. Poult. Sci., v.78, p.1292- 1299, 1999.
• DIBNER, J.J.; BUTTIN, P. Use of organic acids as a model to study the impact of gut microflora on nutrition and metabolism. J. Appl. Poult. Res., v.11, p.453-463, 2002.
• EL-GHANY, W.A.A.; AWAAD, M.H.; NASEF, S.A.; GABER, A.F. Effect of sodium butyrate on Salmonella enteritidis infection in broiler chickens. Asian J. Poult. Sci., v.10, p.104-110, 2016.
• FLORES, F.; LOVATO, M.; WILSMANN, C.G. et al. Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos. Pesqui.Vet. Bras., v.32, p.495-502, 2012.
• GARRIDO, M.N.; SKJERVHEIM, M.; OPPEGAARD, H.; SORUM, H. Acidified Litter Benefits the Intestinal Flora Balance of Broiler Chickens. Appl. Environ. Microbiol., v.70, p.5208-5213, 2004.
• GONZALES, E.; SARTORI, J.R. Aditivos para rações de aves e suínos. Botucatu: FMVZ-UNESP, 2001.
• ISLAM, K.M.S.; SCHUHMACHER, A.; AUPPERLE, H.; GROPP, J.M. Fumaric acid in broiler nutrition: a dose titration study and safety aspects. Int. J. Poult. Sci., v.7, p.903-907, 2008.
• JOZEFIAK, D.; KACZMAREK, S.; BOCHENEK, M.; RUTKOWSKI, A. A note on effect of benzoic acid supplementation on the performance and microbiota population of broiler chickens. J. Anim. Feed Sci., v.16, p.252-256, 2007.
• KHAN, S.H.; IQBAL, J. Recent advances in the role of organic acids in poultry nutrition. J. Appl. Anim. Res., v.44, p.359-369, 2016.
• KHOSRAVINIA, H.; NOURMOHAMMADI, R.; AFZALI, N. Productive performance, gut morphometry, and nutrient digestibility of broiler chicken in response to low and high dietary levels of citric acid. J. Appl. Poult. Res., v.24, p.470-480, 2015.
• LEESON, S.; NAMKUNG, H.; ANTONGIOVANNI, M.; LEE, E.H. Effect of butyric acid on the performance and carcass yield of broiler chickens. Poult. Sci., v.84, p.1418-1422, 2005.
• LILLIE, R.J.; SIZEMORE, J.R.; BIRD, H.R. Environment and stimulation of growth of chicks by antibiotics. Poult. Sci., v.32, p.466-475, 1953.
• PARTANEN, K. Using organic acids in pig feeding as alternative to antibiotic fed additives. In: SIMPÓSIO SOBRE MANEJO E NUTRIÇÃO DE AVES E SUÍNOS, Campinas. Anais... Campinas: CBNA, 2002. p.45-62.
• POLYCARPO, G.V.; ANDRETTA, I.; KIPPER, M. et al. Meta-analytic study of organic acids as an alternative performance-enhancing feed additive to antibiotics for broiler chickens. Poult. Sci., v.96, p.3645-3653, 2017.
• POLYCARPO, G.V.; BURBARELLI, M.F.C.; CARÃO, A.C.P. et al. Effects of lipid sources, lysophospholipids and organic acids in maize-based broiler diets on nutrient balance, liver concentration of fat-soluble vitamins, jejunal microbiota and performance. Br. Poult. Sci., v.57, p.788-798, 2016.
• REZENDE, C.S.M.; MESQUITA, A.J.D.; STRINGHINI, J.H. et al. Ácido acético em rações de frangos de corte experimentalmente contaminadas com Salmonella enteritidis e Salmonella typhimurium. Rev. Bras. Saúde Prod. Anim., v.9, p.516-528, 2008.
• RIBEIRO, R.P.; FLEMMING, J.S.; BACILA, A.R. Uso de leveduras (Saccharomyces cerevisae), Parede celular de leveduras (sscw), ácidos orgânicos e avilamicina na alimentação de frangos de corte. Arch. Vet. Sci., v.13 p.210-217, 2008.
• RICKE, S. Perspectives on the use of organic acids and short chain fatty acids as antimicrobials. Poult. Sci., v.82, p.632-639, 2003.
• ROSTAGNO, H.S.; ALBINO, L.F.T.; DONZELE, J.L. et al. Tabelas brasileiras para aves e suínos: composição de alimentos e exigências nutricionais, 2011. p.141.
• SALAZAR, P.C.R.; ALBUQUERQUE, R.; TAKEARA, P.; TRINDADE NETO, M.A.; ARAÚJO, L.F. Efeito dos ácidos lático e butírico, isolados e associados, sobre o desempenho e morfometria intestinal em frangos de corte. Braz. J. Vet. Res. Anim. Sci., v.45, p.463-471, 2008.
• SAS/STAT® 9.2 user’s guide. Cary: SAS Institute Inc. 2008.
• SHIMOTOYODOME, A.; MEGURO, S.; HASE, T.; TOKIMITSU, I.; SAKATA, T. Short chain fatty acids but not lactate or succinate stimulate mucus release in the rat colon. Comp. Biochem. Physiol., v.125, p.525-531, 2000.
• TREMOCOLDI, W.A.; BRUNINI, O. Caracterização agroclimática das unidades da secretaria de agricultura e abastecimento do estado de São Paulo: Adamantina e Região. Bol. Tec. Inst. Agron., n.204, 24p, 2008.
• VALE, M.M.; MENTEN, J.F.M.; MORAIS, S.C.D.; BRAINER, M.M.A. Mixture of formic and propionic acid as additives in broilers feeds. Sci. Agric., v.61, p.371-375, 2004.
• VAN IMMERSEEL, F.; RUSSELL, J.B.; FLYTHE, M.D. et al. The use of organic acids to combat Salmonella in poultry: a mechanistic explanation of the efficacy. Avian Pathol., v.35, p.182-188, 2006.
• VATTAY, P.; FEIL, W.; KLIMESCH, S.; WENZL, E. et al. Acid stimulated alkaline secretion in the rabbit duodenum is passive and correlates with mucosal damage. Gut, v.29, p.284-290, 1988.
• VIOLA, E.S.; VIEIRA, S.L.; TORRES, C.A.; FREITAS, D.M.; BERRES, J. Desempenho de frangos de corte sob suplementação com ácidos lático, fórmico, acético e fosfórico no alimento ou na água. Rev. Bras. Zootec., v.37, p.296-302, 2008.
• WALDROUP, A.; KANIAWATI, S.; MAUROMOUSTAKOS, A. Performance characteristics and microbiological aspects of broilers fed diets suplemented with organic acids. J. Food Prot., v.58, p.482-489, 1995.
• WILLIAMS, R.B. A compartmentalised model for the estimation of the cost of coccidiosis to the world’s chicken production industry. Int. J. Parasitol., v.29, p.1209-1229, 1999.

Publication Dates

• Publication in this collection
  14 Aug 2020
• Date of issue
  Jul-Aug 2020

History

• Received
  26 Aug 2019
• Accepted
  11 Feb 2020