A blend of benzoic acid and essential oil compounds as an alternative to antibiotic growth promoters in broiler diets

DESCRIPTION OF PROBLEM

Antibiotics have been widely used in animal production for decades. Although some are used therapeutically to improve the health and well-being of animals, most were given for prophylactic purposes and to improve growth rate and feed conversion efficiency (as antimicrobial growth performance promoters, or AGPs) [1]. The mechanism of action of antibiotics as growth promoters is related to interactions with the intestinal microbial population [2]. However, due to the emergence of microbes resistant to antibiotics which are used to treat human and animal infections, the European Commission (EC) decided to phase out, and ultimately ban (January 1, 2006), the marketing and use of antibiotics as growth promoters in feed [1]. The use of antimicrobials as growth promoters in animal nutrition has been questioned in relation to the development of resistant bacteria.

This regulation has forced the countries that are interested in exporting animal products to the European Union to search for alternatives to ensure maximum animal growth without affecting the quality of the final product. In this context, phytogenic feed additives are discussed herein to highlight their growth-promoting effects when supplemented into food animal diets independently, or combined with organic acids. Since the Middle Ages, phytogenic products like essential oils have been widely used for bactericidal, virucidal, fungicidal, antiparasitical, insecticidal, medicinal and cosmetic applications, especially nowadays in the pharmaceutical, sanitary, cosmetic, agricultural and food industries. Also called volatile or ethereal oils, the essential oils are aromatic oily liquids obtained from plant material (flowers, buds, seeds, leaves, twigs, bark, herbs, wood, fruits, and roots) and are complex mixtures of secondary plant metabolites consisting of low-boiling phenylpropenes and terpenes [3, 4].

Organic acids and herbal extracts are 2 important alternatives of great interest to the poultry industry, but the efficacy of organic acids as a replacement for antibiotic growth promoters in broiler chickens has not been adequately demonstrated, however, and relevant information is rather limited [5].

The cumulative effects of essential oils on digestibility of nutrients and on modulation of the gut microflora eventually result in an improvement of broiler performance. Essential oils from different herbs in Turkey were found to improve weight gain, feed conversion, and carcass yield of broilers [6]. A plant extract consisting of 3 EO improved feed conversion and enhanced breast muscle yield in broilers [7].

Organic acids have been shown to have beneficial effects on performance and are widely distributed in nature as normal constituents of plants or animal tissues [2]. They are also formed through microbial fermentation of carbohydrates predominantly in the caeca of poultry [8]. Some (e.g., butyric acid) also decrease the incidence of subclinical necrotic enteritis caused by Clostridium perfringens, an additional beneficial effect which is highly relevant for the poultry industry [9]. Dibner and Buttin [10] stated that dietary supplementation of organic acids resulted in reduction of subclinical clostridial infections in poultry and increased absorption of nutrients, thereby emphasizing their growth-promoting effects. The mechanism of action of organic acids probably reflects their antibacterial nature, such as decreasing the pH of drinking water and reducing the buffering capacity of the feed with subsequent effect on the physiology of the crop and proventriculus [11]. Dibner and Buttin [10] stated that organic acids play a major role in reducing microorganisms such as Escherichia coli, Campylobacter, and Salmonella. These authors affirm that their administration in the diet result in a reduction of subclinical infections in birds, contributing to an increased absorption of nutrients and enhanced digestive and immune systems potentialities.

The CRINA® Poultry Plus product (DSM Nutritional Products Ltd., Basel, Switzerland) is a blend of benzoic acid and essential oil compounds (including thymol, eugenol, and piperine). The objective of this study was to evaluate the addition of CRINA® Poultry Plus in broiler diets and its action as an alternative to antibiotic growth promoters.

MATERIALS AND METHODS

This study was conducted at the Poultry Science Laboratory (LAVIC) in the Department of Animal Science of the Federal University of Santa Maria, Brazil. All procedures used were approved by the Ethics Committee on Animal Use from the Federal University of Santa Maria, Brazil (001/2014) and were carried out in accordance with Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching [12]. A total of 1,550 1-day-old male Cobb 500 broiler chicks from the LAVIC hatchery were used. The birds were vaccinated against Marek’s disease, infectious bursal disease and avian pox virus. The birds used in the study had a range of 42.08 g ± 2.5%.

They were housed in a building with 50 pens with 2.25 m², equipped with a bell drinker, a tray-type feeder for pre-initial phase and a tubular semi-automatic feeder (metal with plastic tray, 20 kg capacity) for the other phases. The heating during the initial phase was made with a 150 W lamp per box. Birds were housed on reused litter to facilitate their exposure to sporulated coccidia oocysts and become infected. Experimental diets were also formulated to contain high levels of crude protein to predispose the chicks to coccidia [13].

The experiment was divided into 3 phases: Initial (1 to 21 d), Growth (22 to 35 d) and Final Phase (36 to 42 d of age). The birds were fed with mash diet (Table 1), the water and feed were provided ad libitum. The diets were formulated according to the Brazilian tables [14] to meet the broiler nutrient requirements and using formulation software for poultry UFFDA.

Experimental Design

The experimental design was completely randomized, with five treatments and ten replicate with 31 birds per pen. The experimental diets were: NC, a basal diet without growth promoters; PC, a basal diet with 10 ppm of Avilamycin (AVI); CPPD, a basal diet with 300 ppm of CRINA® Poultry Plus (CPP) from 1 to 42 d; AVI_(1–21d)/CPP_(22–42d), a basal diet with 10 ppm of AVI from 1 to 21 d and 300 ppm of CPP from 22 to 42 d; and AVI + CPP (1–42 d), a basal diet with 10 ppm of AVI and 300 ppm of CPP from 1 to 42 d. All diets had the same nutrient levels. These diets didn’t have addition of coccidiostats or any type of enzyme.

Body weight gain (BWG), feed intake adjusted by the average number of birds (FI), feed conversion rate (FCR), mortality (MO), and the European productive efficiency index (EPEI) were evaluated in this experiment. FI, BWG, FCR and MO were evaluated in each phase (1-21, 22 to 35, 36 to 42 and 1 to 42 d) and the EPEI was calculated as follows [15]: ADG _(g) * (100 – MO) / FCR * 10.

At 21 d of age, 3 birds per replicate were euthanized (by cervical dislocation, performed by a properly trained professional) to determine the presence and lesion scores of coccidiosis (Eimeria acervulina, E. maxima, and E. tenella). The lesion scores were evaluated according to Johnson and Reid [16]. The development of the broilers intestines was measured by the gut length. At 21 and 42 d, one sample per experimental unit of poultry litter was collected to measure the moisture content. The samples were dried in a laboratory oven at 40°C for 96 h. The incidence of breast and foot pad lesions was evaluated at 42 d of age. The breast lesion scores were evaluated according to Angelo et al. [17] and foot pad lesions according to Martrenchar et al. [18].

Statistical Analysis

Data were subject to analysis of variance using the GLM procedure of SAS [19]. Tukey’s comparison of means test was applied when significant differences occurred at the 0.05 level of significance.

RESULTS AND DISCUSSION

Body weight gain (Table 2) was affected by the inclusion of CRINA® Poultry Plus in broiler diets. Cumulative performance (d 1 to 42) showed that BWG of NC was significantly lower (P < 0.05) than those of PC and CPPD, with CPPD birds having the highest BWG. Furthermore, diets supplemented with CRINA® Poultry Plus and/or Avilamycin improved FCR (P = 0.0001; Table 2) and EPEI (P = 0.0003; Table 3), when compared to NC treatment. No statistical differences between treatments were observed for feed intake (Table 2) except the NC group had higher feed intake (P = 0.0323) than PC, just on the 36- to 42-d period and was not significantly different in relation to the other treatments.

Buchanan et al. [20] obtained positive results in growth performance when phytogenic feed additives were added to a maximum yield diet. FCR was improved in relation to the same diet without the additives, but the BWG was not altered. This last result is not in agreement with the present study. Other studies reported that supplementation with CRINA® Poultry (a blend of essential oils) and other essential oils did not influence the FCR, BW, BWG, and FI results [21, 22, 23, 24].

Synergistic effects of supplementing essential oils combined with organic acids have been reported. For instance, Jang et al. [25] reported that the use of CRINA® Poultry and lactic acid together in broiler feeds improved the final body weight and FCR, when compared to birds fed diets with Antimicrobial Growth Promoters (AGP). Vieira et al. [26] tested the use of a phytogenic feed additive and a blend of organic acids and their interactions. The phytogenic additive or organic acids improved body weight at 21 d and also the cumulative FCR (period of 1 to 42 d) has been improved.

Our results are similar to those found by Weber et al. [27]. These authors carried out a meta-analysis with data from 4 experiments comparing the use of 300 ppm of CPP with non-supplemented controls. They found that CPP significantly improved BW on d 21 and 42. The birds on the CPP treatment showed a higher average daily gain in the starter phase and over the entire experimental period. FCR was better with CPP supplementation than control birds. Mortality was not affected by the dietary treatment. The increase in growth performance of broilers fed diets with CRINA® Poultry Plus found in this study, could be explained by the properties of organic acids and essential oils. The essential oils’ properties have been reported by several authors [3, 22, 28, 29]. They have biological activities as antioxidants and as hypocholesterolemic drugs; they stimulate the animal’s digestive system, increase production of digestive enzymes and improve utilization of digestive products by enhancing liver functions. Essential oils also stimulate blood circulation, reduce the levels of pathogenic bacterias and may enhance the immune status. Moreover, Weber et al. [27] report that essential oils containing piperine significantly reduce the gastrointestinal transit time of feed.

Previous studies confirmed that CRINA® Poultry and other essential oils display antimicrobial activity against intestinal bacteria such as Clostridium perfringens [30], Salmonella typhimurium, and Escherichia coli [31, 32]. It has been reported that this activity is mediated by the lipophilic property that leads to perforation of the bacterial membrane, releasing membrane components from the cells to the external environment [31], and by stabilization of the intestinal microflora and inactivation of C. perfringens toxins [30]. Tampieri et al. [33] studied the efficacy of essential oils against different pathogenic mycetes and found that some essential oils are active against Candida albicans.

Organic acids also have strong antimicrobial activities, can influence mucosal morphology, stimulate pancreatic secretions, serve as substrates in the intermediary metabolism, and improve digestion, absorption and retention of numerous dietary nutrients [34]. Dibner and Buttin [10] reported that after organic acids ingestion, direct antimicrobial activity is highest in the foregut of poultry (crop and gizzard). They improve protein and energy digestibility by reducing microbial competition with the host for nutrients and the endogenous nitrogen losses.

It has been reported that organic acids interfere with cytoplasmic membrane structure and membrane proteins, uncoupling the electron transport and thus reducing ATP production, and disrupt the normal physiology by decreasing the internal pH. The dissipation of proton-motive force and inability to maintain internal pH by bacteria are followed by denaturation of acid-sensitive proteins and DNA [35, 36]. According to Weber et al. [27], blends of various organic acids induced a shift in the intestinal microbiota toward more homogenous and distinct populations and increased Lactobacillus colonization of the chick ileum. Supplemented at 0.1% in broiler diets, benzoic acid particularly increased the dry matter digesta in the ceca; decreased the lactic acid bacteria in the ceca and coliform bacteria populations in the ileum [37]. Kluge et al. [38] reported that benzoic acid has antimicrobial properties, mainly because of its inhibitory effect on several microbial enzymes, in particular α-ketoglutaric acid dehydrogenase and succinic acid dehydrogenase.

Weber et al. [27] report that benzoic acid has been identified as an efficient feed additive to improve growth performance, nutrient digestibility, and nitrogen balance as well as to reduce gram-negative bacteria in the gastrointestinal tract of piglets. Coliform bacteria, however, were decreased in the ceca of chicks, indicating that the considerable antimicrobial activity of benzoic acid could beneficially influence gut health in poultry as well.

In the present study the gut length of the NC group showed lower values (P = 0.0223) than PC and AVI + CPP (1-42 d) (Table 3). By itself, however, CRINA® Poultry Plus did not influence the gut length in comparison to the control group. There were no significant differences in mortality (Table 3) between treatments. These results are similar to those found by Jang et al. [25], Barreto et al. [24], Buchanan et al. [20] and Vieira et al. [26], where mortality, villus height or crypt depth, or intestine weight were not affected by the use of CRINA® Poultry or other phytogenic additives associated or not with organic acids.

Birds fed different treatments had the same lesion scores on breasts and foot pads and litter moisture content (Table 3). These results are similar to the findings of Buchanan et al. [20], as they did not observe significant differences in foot pads.

In the chicken, species of Eimeria develop in different regions in the gut, where, depending on the magnitude of infection, they can cause mild to severe lesions and significant pathology [39]. Thus, E. acervulina develop in the duodenum extending in heavy infections to the mid-intestine, E. maxima develop in the mid-intestine extending to the posterior intestine, and E. tenella develop in the ceca [40].

The scores of lesion by E. acervulina were highest in the NC group birds (P = 0.0050), while the other treatment groups did not show significant differences among them. No significant effects of the treatments were observed for E. maxima and E. tenella lesions (Table 3). Oviedo-Rondón et al. [41] found that the use of CRINA Alternate in broiler diets also significantly reduced coccidiosis lesions in duodenum, but did not affect lesions in the jejunum-ileum. However, our results disagree with the same authors once they found significant reductions of cecal lesion scores with the use of CRINA® Poultry.

Different findings were reported by Oviedo-Rondón et al. [42] who found that CRINA® Poultry and CRINA Alternate did not affect the coccidiosis lesion scores. Giannenas et al. [43] studied the influence of essential oils in broiler diets and found that the lesion scores in the E. tenella-infected group supplemented with essential oils were significantly lower than in the infected control group.

According to Oviedo-Rondón et al. [44], stress and coccidial infection result in drastic shifts in the microbial communities. The use of essential oils modulates the microbial communities in coccidial challenges and avoids drastic changes in the intestinal microbial ecology after a coccidial infection, whether the birds were vaccinated or not.

Hume et al. [45] carried out a study where broilers fed diets supplemented with antibiotic, probiotic, CPP and CRINA® Poultry AF were infected with Eimeria spp. to determine effects of performance enhancers on ileal and cecal microbial communities. They used pyrosequencing and DGGE for sequencing individual cecal bacteria. They also found that probiotics and essential oils blends modified broiler ileal and cecal digestive microbial population. In the final period, however, none of the feed additives reduced the negative effects of infection on BWG to levels seen in non-medicated non-infected controls. Regardless, all feed additives alleviated the coccidian-induced reduction in weight gain. The debilitating effects of Eimeria infection were lessened by these feed additives.

CONCLUSIONS AND APPLICATIONS

1. The supplementation of broiler diets with CRINA® Poultry Plus increased performance and decreased lesion scores of E. acervulina in relation to the broilers in the control group.
2. The diet with inclusion of only this additive showed better results in body weight gain than the positive control diet.
3. CRINA® Poultry Plus can be used as an alternative product to antibiotic growth promoters in broiler diets without losses in productive performance.