In recent years, use of probiotics, prebiotics and synbiotics those that enrich certain bacterial population in the digestive system are considered as alternatives to antibiotic growth promoters in poultry nutrition (Patterson and Burkholder, 2009) pertaining to their potential to reduce chances of infection in poultry and subsequent contamination to poultry products. Fuller (1989) defined probiotics as “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. It consists of live microbial cultures that are isolated from the gastro intestinal tract (GIT) of a healthy adult animal of the same species to which probiotics will be given, as it is usually species specific (O’Dea et al., 2006). These beneficial microflora are known to increase the host’s protective barrier against enteric bacteria, humoral immune reaction against pathogens and cell immunity response (Taheri et al., 2014).  Lactobacilli, also considered as beneficial bacteria, were found to be the most prominent bacteria in the crop of birds after the first week of age (Frei et al., 2001). The predominant species of Lactobacillus are L. reuteri, L. salivarius and L. animalis (Vineetha, 2014). Prebiotics are selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well-being and health (Gibson et al., 2004). Mainly prebiotics are small fragments of carbohydrates and commercially available as oligosaccharides of galactose, fructose or mannose (Ganguly, 2015). Currently, combined administration of probiotics and prebiotics referred to as synbiotic, is gaining momentum in poultry production for its beneficial effects.

Assam local chickens reared under natural conditions without much intervention of modern day management and feeding practices adapt well to local climate and possesses disease resistance capacity (Kalita et al., 2009). These special attributes of the local chickens may have been influenced by beneficial microflora like the Lactobacilli present in the gut boosting its immune system. Thus, these microbes when isolated and characterized from the gut of local chicken may prove useful as probiotic culture for commercial exploitation. Subsequently, Lactobacillus reuteri PIA16 was isolated from the gut of Assam local chicken and characterized at Institute of Microbial Technology (IMTECH), Chandigarh. To evaluate if Lactobacillus reuteri PIA16 was a putative probiotic, its effect on the productive performance of broilers and any synergic effect when supplemented with prebiotic were studied.

Materials and Methods

Two strains of Lactobacillus isolated from different gut region i.e., ACE5 (caecum) and AJ3 (jejunum) both showed 99.72 % genetic identity with Lactobacillus reuteri through the 16S rRNA gene sequence and BLAST search analysis conducted under the Institute of Microbial Technology (IMTECH), Chandigarh, India. The strain is registered as Lactobacillus reuteri PIA16 (accession No. KX260961) under GenBank, National Centre for Biotechnology Information (NCBI), India. These two strains were used for in-vivo growth bioassay as two separate entities so as to also examine any variation between the strains pertaining to the different gut regions they were harvested from. As premix, 20% of daily ration required for broilers was autoclaved (Patra, 2013; Vineetha, 2014) and daily inoculated with 20% of ACE5 and AJ3 broth culture separately and fermented at 37°C for 48 hrs. The fermented feed was added daily to the required daily ration and mixed well. The initial average count of the isolates ACE5 and AJ3 were 1.04×10⁸ cfu/ml and 1.05×10⁸ cfu/ml, respectively but after fermentation the count of Lactobacillus increased to 1.85×10⁸ cfu/gm and 1.89×10⁸ cfu/gm of fermented feed for ACE5 and AJ3, respectively. The commercially available prebiotic, mannan oligosaccharide (MOS) @ 0.25% (Kim et al., 2011) was used for the experiment.

A total of 240 day-old chick broilers (Cobb) were randomly assigned into five groups of 48 birds containing six numbers in each of eight replicates of mixed sex per group.  They were provided with different dietary treatments i.e. a control -basal diet (T₁), T₁ + ACE5 at 1.85×10⁸cfu fermented feed (T₂), T₁ + AJ3 at 1.89×10⁸cfu fermented feed (T₃), T₂ +ACE5-MOS (T₄) and T₃ +AJ3-MOS (T₅) at 0.25% of the total feed. The birds were housed in battery cages and reared under standard managemental conditions for duration of 5 weeks. The growth parameters were recorded weekly. Daily mortality, if any, was recorded for estimation of livability % during 0-3 and 4-5 weeks. Broiler Performance Efficiency Index (BPEI) and the cost of production per kg live broiler were calculated according to the formula described by Narahari and Kumararaj (2008) to evaluate the economy of feeding Lactobacillus reuteri PIA16 and MOS in broilers. Data obtained were statistically analyzed with one way Analysis of Variance and Duncan’s multiple range tests using the software SAS System (Local, X64_7PRO).

Results and Discussion

Growth Performance                                                                            

Difference in the body weight change among the groups were pronounced by 3^rd week where all the Lactobacillus reuteri PIA16 fed groups showed significant (P<0.05) improvement in body weight compared to control (Table 1). The MOS supplemented groups showed significantly better result than T₂ and T₃ and most superior was the T₅ group. The body weight change in the remaining weeks also followed the same trend. This growth promoting effect of dietary Lactobacillus supplementation and prebiotic in broiler diet were also noted in several reports (Murry et al., 2006; Zhu et al., 2009; Sherief and Sherief, 2011; Saiyed et al., 2015). The improvement observed in the study maybe due to the improvement of the intestinal environment with the presence of probiotic bacteria, thus, increasing the efficiency of digestion and nutrient absorption processes (Alkhalf et al., 2010). Moreover, prebiotic are known to play a role in augmenting the activity and/or the composition of the probiotic bacteria, thereby, improving the performance of the broilers (Gibson et al., 2004).

All the treated groups showed significantly (P<0.05) better weight gain than control group. A noticeable increase (P<0.05) in body weight gain in groups supplemented with MOS compared to their respective counterparts were observed in the last two weeks. An increase in daily weight gain to the extent of 14 per cent by 4-day application of lactic acid bacteria culture was observed (Strompfova et al., 2005). Similar reports also revealed significantly higher weight gain in the broilers fed with probiotic cultures containing Lactobacillus (Swamy and Upendra, 2013; Song et al., 2014). Better overall performance of the broilers fed with Lactobacillus and prebiotic in terms of feed efficiency and growth in the present study might be due to the improvement of the viability of the probiotic bacteria through the usage of the prebiotic as a substrate for fermentation by the probiotic bacteria which subsequently produced lactic acid, reducing further the pH of the GI tract and have suppressive effect on unwanted organisms and thus, controls the population of pathogenic micro-organisms,  promoting the health status of the broilers (Bansal et al., 2011; Falaki et al., 2011).

Table 1: Growth performance of broiler chickens fed with Lactobacillus reuteri PIA16

^abcMeans bearing same superscripts in a row do not differ significantly (P≤0.05)

The feed consumption of the broilers which consumed L. reuteri PIA16 in their ration were comparatively more than control birds by 3^rd week but little difference was observed among the treated groups (Table 1). Similar finding was also reported by Falaki et al. (2011) on using synbiotic in broiler chickens. Total feed consumption per bird was recorded highest in group T₅ (2575.99 g) followed by group T₄ (2556.17 g), though there were no pronounced difference with the remaining groups. The improvement in feed intake may be accounted for the improved growth performance of the broilers fed with dietary supplementation of Lactobacillus and prebiotic resulting from the stimulation of favourable microbial balance in the gut and consequently improve the efficiency of digestion and nutrient absorption process of the host. In a more recent study, an overall increase in feed intake in chicken fed with multistrain probiotics than the control group was reported (Zhang and Kim, 2014).

A better FCR in T₄ and T₅ groups was observed than T₂ and T₃ groups while the least was recorded in T₁ group in the 1^st week (Table 1). Same trend was observed in the following weeks and the groups supplemented with probiotic and prebiotic showed an augmented effect on FCR when compared with groups fed only with probiotic. This may be attributed to the synergism between probiotic and prebiotic followed by better absorption of nutrients by the GI system which is responsible for better feed conversion efficiency. Overall, T₅ group showed the best FCR. Broilers supplemented with L. plantarum and L. reuteri Pg4 were reported with improved FCR (Vandeplas et al., 2009; Yu et al., 2007). Using probiotic culture containing Lactobacillus indicated lowered FCR in broiler chickens (Alkhalf et al., 2010; Murry et al., 2006).

Economics of Production

No mortality was recorded during 0-5 weeks showing 100% livability in all the groups. The Broiler Performance Efficiency Index (BPEI) indicated T₅ (109.09) group to be most efficient followed by T₄ (105.16), T₃ (96.89)_, T₂ (96.30) and T₁ (83.83). The cost of production per bird (Rs.) was recorded highest in T₅ (136.86) group and lowest in T₁ (127.52) group with the remaining groups falling in between the range. The higher production cost in T₅ and T₄ might be due to higher feed consumption which resulted in higher body weight. Despite higher production cost in the Lactobacillus fed groups, the gross profit per broiler (Rs.) was recorded highest in T₅ (31.14) followed by T₄ (28.53), T₃ (22.17) and T₂ (19.51) while lowest gross profit was recorded in T₁ (12.48) notwithstanding the low production cost. Feeding cost in probiotic fed broiler chickens was found lower (Anjum et al., 2005; Doley, 2011) and higher profit on using synbiotic (Saiyed et al., 2015) were reported. An improvement in the economic efficiency of the synbiotic fed broilers was also eminent (Narasimha et al., 2015). The high gross profit in all the Lactobacillus fed groups might be because of the higher body weight which generated economic benefit.

Conclusion

Better overall productive performances were observed in the broilers which received dietary L. reuteri PIA16 at 10⁸cfu level along with mannan oligosaccharide. L. reuteri PIA16 can be a promising probiotic strain whose beneficial effect can be improved further when supplemented along with prebiotic, mannan oligosaccharide. The study also revealed no variation between the Lactobacilli showing singularity and non site-specificity. Furthermore, feeding dietary L. reuteri PIA16 with MOS to commercial broiler chickens generated high gross profit despite higher production cost. The Broiler Performance Efficiency Index (BPEI) reflected an increase in the index in all the Lactobacillus fed groups showing a positive impact on the economy.

Acknowledgement

The authors acknowledge the Department of Biotechnology, Ministry of Science and Technology, the Government of India for funding the entire research work.

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