Probiotic and synbiotic in broiler diet: performance and Enterobacteriaceae

The objective of this study was to evaluate the effects of probiotics and synbiotics on the performance and Enterobacteriaceae count of broiler chickens. A total of 640 one-day-old male broiler chicks were distributed in a completely randomized design with four treatments and eight replicates with 20 birds each. The treatments were: ration with performance enhancer (zinc bacitracin; positive control); ration without performance enhancer and probiotic/synbiotic (negative control); ration with probiotics; and ration with synbiotics. At 35 days, five birds from each treatment were euthanized and intestinal contents were harvested for determining the Enterobacteriaceae count. The performance data and average colonyforming units (CFUs) transformed as log CFU/g were subjected to analysis of variance and Tukey’s test. The effects of probiotics and synbiotics were observed in the initial phase, with supplemented birds exhibiting comparable weight gain to those supplemented with bacitracin. No effect of the treatment on broiler performance was observed after 42 days. The enterobacterial count was comparable among all experimental treatments. Supplementation with probiotics and synbiotics did not compromise the performance of broilers and did not alter the Enterobacteriaceae count.


INTRODUCTION
The use of antimicrobials has provided better feed conversion and weight gain in poultry for more Arq. Bras. Med. Vet. Zootec., v.72, n.6, p.2365-2372, 2020 Colistin sulfate was banned in 2016, followed by a more recent prohibition of tylosin, lincomycin, and tiamulin, by the Brazilian Ministry of Agriculture.
Thus, with the decreasing number of permitted antimicrobials, it has become necessary to study alternative additives that can achieve similar results, maintain intestinal health and immune system of birds, and improve nutrient utilization without inducing bacterial resistance, particularly probiotics and prebiotics . Probiotics consist of one or more live microorganisms that beneficially affect the endogenous microbiota and may include species of Lactobacillus, Bifidobacterium, Enterococcus, and yeast (Al-Khalaifa et al., 2019), whereas prebiotics are defined as ingredients that are nondigestible by animals and serve as substrates for beneficial bacteria. The combination of probiotics and prebiotics yields is called synbiotics, e.g., fructooligosaccharides (FOS) combined with bifidobacterial, which exert a synergistic effect and enhance bird performance, without leaving residues in the carcass (Aziz-Mousavi et al., 2015;Beski and Al-Sardary, 2015;Reis and Vieites, 2019).
The addition of probiotics to chicken feed ameliorates feed conversion and body weight, which may be attributed to higher absorption of nutrients and improved bird performance and immunity, as reported by Royan (2018) using the probiotic species Enterococcus faecium. He et al. (2019) reported better performance and greater production of immunoglobulins in broilers fed diets containing Bacillus subtilis. Similarly, Ramlucken et al. (2020) reported that B. subtilis and Bacillus velezensis produce exogenous enzymes, improving nutrient utilization and feed conversion in supplemented animals.
Similar to probiotics, synbiotics can improve nutrient absorption because of the contained prebiotics, as stated by Aziz-Mousavi et al. (2015), who used a synbiotic combination of E. faecium and fructooligosaccharides in chicken feed and noted greater weight gain, better feed conversion, and higher protein consumption in supplemented birds. Cheng et al. (2017), Shokri et al. (2017), andKridtayopas et al. (2019) also reported better bird performance on addition of synbiotics to chicken diet.
In addition, probiotics and synbiotics promote multiplication of beneficial bacteria to the detriment of pathogenic bacteria, as demonstrated by Fonseca et al. (2010) and Zhang et al. (2014). Śliżewska et al. (2020) reported higher numbers of Bifidobacterium spp. and Lactobacillus spp., and lower numbers of Clostridium spp. and Escherichia coli in the intestine of broilers supplemented with synbiotics. Kridtayopas et al. (2019), Mora et al. (2019), and Chen and Yu (2020) also reported an improvement in the population of beneficial bacteria along with pathogen control by synbiotics. Ateya et al. (2019) observed that synbiotics combined with organic acids weakened the inflammatory response in the intestine of chickens challenged with E. coli, resulting in better performance, and lower mortality and bacterial elimination in the environment. In summary, replacement of performance enhancers by proand synbiotics has varying effects on the performance and intestinal microbiota of poultry. The best response depends on several factors, such as product composition, microorganism survival, dosage, and the challenge or stress conditions to which birds are exposed (Reis and Vieites, 2019;Shanmugasundaram et al., 2019;Chen and Yu, 2020). Therefore, the objective of this study was to determine the effects of probiotic and synbiotics on the performance and Enterobacteriaceae count of chickens challenged with reused litter and untreated water.

MATERIAL AND METHODS
The experiment was approved by the Animal Use Ethics Commission (CEUA)-IF Goiano, under protocol number 7572161018. Experiments were performed using 640 one-day-old male Cobb broiler chicks (40.0 ± 4.0g) distributed in four treatment groups, with eight replicates each and 20 birds per replicate, following a completely randomized design. The experimental diets (Table 1) were formulated according to the recommendations of Rostagno et al. (2017) based on the nutritional requirements during the different breeding phases, and consisted of prestarter (1 to 7 days), starter (8 to 21 days),grower (22 to 35 days), and finisher (36 to 42 days) diets.
The addition of the probiotics or synbiotics were added based on the manufacturer's recommendations, and the treatments were designed as follows: Treatment 1 -feed with performance enhancer (zinc bacitracin; positive control); Treatment 2 -feed without performance enhancer and probiotic\synbiotic (negative control); Treatment 3 -feed supplemented with probiotic (150g/ton feed). Treatment 4 -feed supplemented with synbiotic (100g/ton of feed).

RESULTS AND DISCUSSION
The diets with the addition of probiotics and synbiotics provided broilers with similar weight gain and final weight results as those fed diets containing zinc bacitracin (Table 2). It is thus evident that the alternative additives did not compromise the weight of broilers in the starter phase, as opposed to broilers that did not receive any additives, which presented lower body weight. This is consistent with the results of Regarding water quality, it is common practice to provide chlorinated water and to clean pendular drinkers at least twice every day. Amoroso et al. The better performance of birds in the starter phase may be attributed to improved nutrient utilization on supplementation with probiotics and synbiotics because some microorganisms used as probiotics, such as Bacillus spp., produce exogenous enzymes that promote degradation and more efficient use of nutrients. This was reflected in the higher feed conversion and greater weight gain of birds at 21 days of age, which was consistent with the results of He et al. (2019) and Ramlucken et al. (2020). During the entire breeding period, i.e., from day 1 to 42 (Table 3), the performance of birds fed diets without antimicrobial supplementation (Treatment 2) and with added probiotics and synbiotics (Treatments 3 and 4) was comparable to that of birds supplemented with a conventional antimicrobial (zinc bacitracin; Treatment 1). to which birds were subjected may not have been sufficient to verify the beneficial effects of probiotics. However, birds were exposed to a more intense sanitary challenge in the present study, in the form of reused litter without treatment and cleaning of the pendular drinkers only once per week, which resulted in lower weight and poor feed conversion after 42 days.
Mora et al. (2019) did not observe any effect of synbiotic administration via water on feed conversion and weight gain of broilers challenged with Salmonella Typhimurium and Clostridium perfringens after 42 days. The authors attributed the differences in the results of probiotic-and synbiotic supplementation to the condition of the experimental birds, species included in the probiotic, and the type of fiber contained in the prebiotic.
In general, our study presented satisfactory results for probiotics and synbiotic supplementation. During both the starter phase and the total breeding period, the performance of broilers supplemented with the alternative additives was comparable to that of birds that received zinc bacitracin, which is a traditional antimicrobial for poultry. It should be noted that when considering the performance of birds supplemented with probiotics or synbiotics, several factors, such as probiotic composition, dosage, route of administration, and type and viability of microorganisms in the gastrointestinal tract, must be taken into account in addition to the imposed health challenge, as emphasized by Mora et al. The treatments did not affect the total Enterobacteriaceae count in the small intestine (Table 4) In addition, birds subjected to the sanitary challenge likely ingested a greater amount of coliforms, because of the poor quality of the water provided (without chlorine and containing organic matter and/or excreta because of unclean drinkers). According to Amoroso et al. (2015), untreated and poor-quality water contains a higher number of coliforms, including E. coli. However, the additives did not alter the Enterobacteriaceae population, which was consistent with the observations of Sherief et al. (2012), who did not report any effect of probiotics and synbiotics on the total count of coliforms, Escherichia coli, or aerobic bacteria. Our results were also in agreement with those of Torok et al. In contrast, the use of probiotics or synbiotics has been reported to alter the microbiota in terms of the numbers of beneficial gram-positive bacteria, such as Lactobacillus sp. and Bacillus sp., which were shown to multiply and produce acids, thus lowering the intestinal pH and likely controlling pathogenic microorganisms (Sherief et al., 2012). Chen and Yu (2020) analyzed the fecal microbiota of chickens supplemented with Bacillus licheniformis and noted a greater amount of Lactobacillus on probiotic addition to feed (3g/kg), demonstrating a positive correlation between the additive and higher body weight of birds.
Synbiotic supplementation significantly increased the lactic acid level and total short chain fatty acids (i.e., acetic, propionic, valeric, butyric, and formic acids). Short chain fatty acids play important roles in pH regulation, increase calcium, iron, and magnesium absorption, and positively affect hepatic metabolism of glucose and proteins. These fatty acids are very important in the maintenance of the structure, function, and integrity of the intestine for inhibiting Salmonella, E. coli, and Campylobacter (Śliżewska et al., 2020).
According to He et al. (2019) and Ramlucken et al. (2020), the probiotic composition is important because certain bacteria, such as B. subtilis, B. velezensis, and B. licheniformis, are aerobic and provide a suitable environment for lactobacilli and bifidobacteria under anaerobic conditions, which subsequently produce acids and lower the intestinal pH, preventing the growth of pathogenic microorganisms. Bacillus, Lactobacillus, and Bifidobacterium aregram-positivegenera and were not investigated in the present study because the culture medium used, i.e., MacConkey agar, specifically selects gram-negative bacteria.
The results of the present study and those of other studies on the effect of additives on enteric microbiota evidence the microorganismal diversity and indicate that changes in the enterobacterial population depend on multiple factors. Thus, the handling of litter, food offered, type and composition of the probiotics\synbiotics, method of enterobacterial determination, included microorganisms, and intestinal location for the Enterobacteriaceae count must be considered. Furthermore, it is important to test new probiotic strains, including some fungal species, for potential poultry applications.

CONCLUSION
The addition of probiotics and synbiotics did not compromise the performance of broilers and did not alter the intestinal Enterobacteriaceae count.