Synergistic effect of non starch polysaccharide enzymes, synbiotics and phytase on performance, nutrient utilization and gut health in broilers fed with sub-optimal energy diets

Aim: In view of the ban on antibiotic growth promoters (AGPs), an attempt was made to explore the possibility of harnessing synergistic effect of non starch polysaccharide (NSP) enzymes, synbiotics and phytase on performance, nutrient retention, gut health and histology of broilers fed with corn-soybean meal based low calorie diets. Materials and Methods: A total of 150 a day-old broiler chicks were weighed, wing banded and randomly distributed into five experimental groups, six replicates per group and five birds per replicate and raised in electrically heated battery brooders. Evaluated the synergistic effect of the NSP enzymes, synbiotics and phytase alone or in combination, supplemented to cornsoybean meal based broiler diet, low in energy concentration (Basal diet (BD)) (-225 kcal lower metabolizable energy than standard diet (SD), on performance, nutrient retention, carcass traits, gut conditions and cost per kg live weight gain. Results: The body weight gain in broiler chicks fed with BD supplemented with NSP enzymes, synbiotics and phytase was significantly (P<0.01) higher. Supplementation of NSP enzymes, synbiotics and phytase alone or in combination had significant effect on feed intake. Synergistic effect of NSP enzyme, synbiotics and phytase was observed on overall feed conversion ratio (1.86), which improved (P<0.05) in comparison to BD (2.06) and SD (2.02), respectively. The supplementation of NSP enzymes, synbiotics and phytase to BD improved (P<0.05) utilization of organic matter (OM), crude protein (CP), nitrogen free extract (NFE), gross energy (GE), phosphorus and the tibia ash compared to BD, whereas no effect on retention of DM and CF was observed. Intestinal viscosity and E. coli count significantly (P<0.01) reduced with addition of NSP enzymes, synbiotics plus phytase or combination of all. The supplementation of NSP enzymes, synbiotics plus phytase had no effect on intestinal histology. The cost of feeding was lower (P<0.01) in BD. Addition of these feed additives to BD did not increase the feeding cost and was comparable to unsupplemented ones and lower (P<0.01) than SD. Similarly, the feed cost per kg live weight gain during various phases of broiler production was reduced (P<0.01) due to supplementation of all the feed additives compared to SD and BD. Conclusions: It can be concluded from the above experiment that supplementing sub-optimal energy diets with NSP enzymes along with synbiotics and phytase improved body weight gain, FCR, nutrient retention, tibia ash and reduced the cost of production considerably.


Introduction
In view of the above circumstances, there has long been interest in finding alternatives to antibiotics for poultry The productivity of broilers has improved production. Resident microbes in the birds' digestive significantly, through genetic improvements. Increased tract have a profound effect on some of the physiolorearing density has concentrated and increased disease gical processes of their host. It is important to understand challenges making birds more susceptible to various the dynamics of the intestinal microbial ecology of the pathogens especially enteropathic microbes such as chicken to find alternatives to antibiotics. Under normal Escherichia coli, Salmonella spp., Clostridium circumstances there is a delicate balance of beneficial perfringens and Campyobacter spp. This increased and pathogenic bacteria in the gastrointestinal tract susceptibility has resulted in the use of antimicrobial (GIT). This is influenced by symbiotic and competitive growth promoters which are primarily used to enhance interactions and relationships. The microbial communities gut health and control sub-clinical challenges. With will not only protect the GIT but also enhance increasing public health concerns about bacterial productivity in the host. NSP enzymes degrade NSP resistance to antibiotics, the use of antibiotics in and by this improve gut motility and nutrient (mainly therapeutic or sub-therapeutic doses in poultry feed has energy) availability [1]. Prebiotics are non-digestible been severely limited or eliminated in many countries.
substances, mainly oligo-and polysaccharides, lowering pH in the gut and by this inhibit colonization of pathogenic microorganisms, stimulate immunity and neutrale toxins. Probiotics act by competitive exclusion, lower gut pH, produce bacteriocins, lysozyme and peroxides, and stimulate the immune system. The after approval of Institutional Animal Ethics Committee. combined application of prebiotics and probiotics is Experimental design and sample collection: One called synbiotics [2]. Feed additives if incorporated in hundred and fifty (150) day-old Cobb commercial poultry feeds, can create favourable conditions in the broiler chicks were weighed, wing banded and intestine for the efficient digestion of feed [3,4]. Many randomly distributed into five experimental groups, six feed additives viz., NSP enzymes, prebiotics, probiotics, replicates per group and five birds per replicate. The acidifier more or less help in maintaining gut directly or NSP enzymes combination (xylanase 7500 IU/kg, indirectly. In most of the experiments these additives cellulase 100 IU/kg and β-D-glucanase 100 IU/kg), have been used singly.If two or more such additives are prebiotic (MOS, 0.5g/kg), probiotic (Saccharomyces 8 used in combination, possibly their effects may compboullardii, 10 CFU/kg) and phytase (675 IU/kg) was lement and may have synergistic effect.
tested at sub-optimal energy concentration (225 Keeping these objectives in mind the present kcal/kg less ME than standard diet) [5]. The details of study was conducted to exploit the synergistic effect of experimental diets are given in Table 1, 2 and 3. All NSP enzymes, prebiotics, probiotics (synbiotics) and replicate groups of chicks were offered the respective phytase, on performance, nutrient retention, gut health diets ad libitum for a period of 42 days. Weekly body weights and feed intake were recorded. At the end of and histology. experiment, a metabolic trial of 4 day duration was

Materials and Methods
conducted to determine the nutrient utilization and Ethical approval: This research work was carried out balance of nutrients. The samples of each feed, feed  residue and feces pooled during 4 days period were standard and basal diets is presented in Table-4. ground and analyzed for proximate principles as per Body weight gain (g): The body weight gain in broiler method described previously [6]. After metabolic trial, chicks fed with BD supplemented with NSP enzymes, 30 birds comprising of 6 birds from each diet by synbiotics and phytase is presented in Table-5. selecting one at random from each replicate) were Significantly higher (P<0.01) weight gains were slaughtered to assess the carcass characteristics. st observed during 1 week in broiler chicks fed with Gut health: To study the effect of dietary energy basal diet (BD) supplemented with synbiotics and concentration, supplementary effect of NSP enzymes, phytase (80.83g) or BD supplemented with NSP synbiotics and phytase on gut health, the digesta was enzymes, synbiotics and phytase (81.47g) compared to collected from distal portion of small intestine during BD (63.50g) and SD (68.57g). Starter phase, finisher slaughter. Approximately two grams of digesta was phase and total period (1-42 d) no difference in weight taken in sterile eppendorf tubes for enumeration of E.
gain was observed among the chicks fed with SD, BD, coli. Another 2 g of digesta was collected and BD supplemented with NSP enzymes, BD centrifuged at 5000 rpm for 10 minutes at 20 °C. An supplemented with synbiotics and phytase or in aliquot of supernatant (0.5 to 1 ml) was collected and combination of synbiotics, phytase and NSP enzymes. stored in capped vials for viscosity determination. The Feed intake (g/bird/day): The feed intake by chicks digesta collected in centrifuge tubes was utilized for fed SD during starter phase was higher (P<0.01) than measuring the pH.
BD, BD supplemented with NSP enzymes, BD Histology of intestines: Representative pieces of duodenum supplemented with synbiotics and phytase or BD in of intestine were collected in 10% formal saline and combination of all feed additives fed chicks (Table-5). preserved for histological studies. After proper fixation Supplementation of NSP enzymes, synbiotics and the intestinal tissue was trimmed and subjected to over phytase alone or in combination had significant effect night washing, dehydration in various percentages of on feed intake. Supplementation of NSP enzymes or alcohol, cleaning in xylol, embedding in paraffin wax synbiotics and phytase alone or in combination of all for preparation of blocks [7]. The paraffin blocks were above feed additives to BD reduced (P<0.01) the feed cut into 5µ thick sections and stained with routine H & intake. The starter phase feed intake was higher E stain [8] and used for microscopic examination.
(P<0.01) in BD fed chicks than SD and comparable to BD supplemented with NSP enzymes. The addition of Statistical analysis: The data were subjected to NSP enzymes, synbiotics and phytase to BD reduced statistical analysis using Statistical Package for Social th (P<0.001) the feed intake in chicks but was higher than Sciences (SPSS) 16 version and comparison of means those fed SD. During finisher phase the feed intake by was tested using Duncan's multiple range tests [9].
BD fed chicks was lower (P<0.001) than SD fed chicks Results and addition of feed additives alone or in combination Nutrient composition of experimental ration: Nutrient had no effect on feed intake. While overall feed intake composition (% dry matter basis) of broiler finisher was higher (P<0.001) in SD but comparable to BD and  compared to SD (31.14%), BD (31.59%), BD The FCR of BD fed chicks during starter phase was supplemented with NSP enzymes (32.70%). However higher (P<0.001) than those fed SD. Supplementing the phosphorus retention was comparable between BD with NSP enzymes or synbiotics and phytase or synbiotics group and BD supplemented with NSP combination of all feed additives improved the FCR in enzymes, synbiotics and phytase. The tibia ash content starter phase and comparable to SD. During finisher was lower in BD (42.74%) compared to SD (46.39). phase and overall period, the FCR was comparable Supplementing BD with NSP enzymes or with between SD and BD. Synergistic effect of NSP synbiotics and phytase or combination of synbiotics, enzymes, synbiotics and phytase was observed for phytase and NSP enzymes improved the tibia ash FCR during finisher phase (1.99) and overall period content and was comparable to SD (Table-6 (Table-5). The starter, finisher and total viscosity was higher in SD compared to BD. The E. coli weight gains recorded was though comparable among count (log ) in intestinal contents was lowest 10 various groups, higher weight gain of 7.48% was (P<0.001) in SD (4.34) compared to BD (6.68) (Table  recorded with supplementation of all feed additives to 6). Supplementation of synbiotics with phytase (1.32) BD. The results are in agreement with [10,11] who or combination of all feed additives (1.97) lowered the reported improvement in weight gains with supple-E. coli count in intestinal contents in comparison to BD mentation of various feed additives (avilamycin, and SD. allzyme, avimos, biomos, yeast extract, xylanase, avizyme and gustor) individually or in combination. Gut histology: The supplementation of NSP enzymes Improvement in live weight gain was also reported [12] along with synbiotics and phytase did not influence the when broiler diets supplemented with direct fed intestinal histology, except few broad elongated and microbials (DFM), antibiotic growth promoters (AGP) folded, congested villi with presence of few goblet and Biomos compared to control. However, contrary to cells was noted (Figure-1).
present findings, addition of prebiotics (galacto oligosacharides) and probiotics (Bifiodobacteriam 9 lactic 300x10 cells/g) individually or in combination had no effect on body weight gain [13].
Feed intake: During starter period feed intake (g/d) was higher (P<0.05) in BD compared to SD and BD supplemented with feed additives (Table-5). However, significantly higher (P<0.01) feed intake was recorded in SD fed birds during finisher period compared to BD and BD supplemented with various feed additives. (Avilamycin, Allzyme, Avimos, Biomos, Yeast extract, Cost comparison: The cost of production per kg live xylanase, Avizyme and Gustor) individually and weight gain during starter and finisher phases of broiler combination had no effect on DM intake over the entire production is given in Table-5. The cost of production 1-28 day experimental period. Mean feed intake for the per kg live weight gain during starter phase was higher whole period was numerically greatest for the birds fed (P<0.01) for BD (Rs. 34.08) compared to SD (Rs. on positive control. The average daily feed intake 31.55). Supplementing BD with NSP enzymes (Rs. (ADFI) of birds fed with direct fed microbials (DFM), 31.74) or synbiotics alongwith phytase (Rs. 31.77) antibiotic growth promoters (AGP) and Biomos was reduced the cost of production per kg weight gain insignificant compared to control [12]. It was found no compared to BD, but was similar to SD. Addition of all effect of feed additives (xylanase, protease and the feed additives to BD increased the feed cost (Rs. phytase) when supplemented to nutritionally marginal 35.92) compared to all other groups during starter (2870 kcal of ME/kg, 0.85% Ca and 0.24% available P) phase. During finisher phase, cost of production was corn soy bean based diets on feed intake [14]. However, a highest (P<0.01) for SD (Rs. 40.50) but comparable study [11] reported that diets supplemented with with BD (Rs. 39.57). Supplementation of NSP performance enhancers (prebiotics, probiotics and enzymes, synbiotics along with phytase or all feed organic acids either alone or in combination) had additives reduced (P<0.01) the cost of production significantly influenced feed intake for 21 day compared to SD during finisher phase. Similarly the (P<0.05) period but not at 0 to 42 day period. overall cost of production was similar between SD and BD. Feeding of BD containing either NSP enzymes Feed conversion ratio: Birds fed with BD had poorer (P<0.05) feed conversion efficiency compared with breast yield per cent than SD and BD (Table 7). those fed with SD. Supplementation of NSP enzymes, Similarly, dietary treatments had no effect on synbiotics and phytase and combination of all abdominal fat pad weight, liver and gizzard. improved feed conversion efficiency linearly indicating There was no effect of biomas, protexin and synergistic effect of addition of two or more feed acidifier individually or combination of all on edible additives to low calorie diets (Table-5). Earlier it was carcass yield, liver weight and gizzard weight [20]. reported that significant improvement in FCR with However, on the other hand another study [21] reported addition of various feed additives (avilamycin, improved dressing yield per cent (P<0.05) and breast allzyme, avimos, biomass, yeast extract, avizyme and yield per cent and other meat yield traits when gustor), combination of prebiotics and probiotics supplemented with or without growth promoters compared to control and xylanase, protease, amylase (flavomycin, avilamycin, genex and avila m/z) and and phytase to nutritionally marginal (2870 kcal of were comparable (P>0.05) among the groups. ME/kg, 0.85% Ca and 0.24% available P) corn soy Gut conditions: Supplementation of NSP enzymes, bean based diets [10,11,14]. However another study synbiotics and phytase alone and combination had no [15] observed that addition of xylanase, amylase, effect on intestinal pH values recorded among the protease and phytase alone to low density diets had no treatment groups (Table-6). Whereas viscosity and E. effect on feed conversion ratio, but combination had coli count significantly (P<0.01) reduced in feed significantly (P<0.05) improved feed efficiency additives supplemented groups compared to SD and compared to the negative control.
BD. The intestinal pH recorded were in agreement with Nutrient retention: The supplementation of NSP earlier findings [20] that observed supplementing enzymes, synbiotics and phytase alone or in prebiotics, probiotics and acidifier singly or in combination of all improved the retention of OM, CP, combination had no effect on pH. Viscosity recorded NFE, GE and phosphorus (Table 6). Whereas DM, CF was in agreement with the earlier report [22] which and EE retentions were not influenced by dietary indicated that the exogenous enzyme supplementation treatments compared to BD and SD. BD with significantly (P<0.05) reduced the digesta viscosity. synbiotics and phytase influenced the retention of OM, Previously significant effect on lactobacilli and CP, CF and NFE. On the whole, the addition of NSP coliform counts in ileum of the diets supplemented enzymes, synbiotics and phytase to BD improved with different feed additives alone or in combination (P<0.05) retention of these nutrients significantly. The were reported [10]. results are in agreement with earlier report on Gut histology: The supplementation of NSP enzymes improvement in AME and phosphorous retention by with synbiotics and phytase to BD resulted in broad supplementation of phytase alone or in combination villi at tip with distinct goblet cell activity ( Figure 1). with NSP degrading enzymes and citric acid [16].
The effect of synbiotics (BIOMIN IMBO) increased (P Supplementation of exogenous enzymes to the < 0.001) villus height/crypt depth ratio and villus broiler diet improved starch digestibility and consequently DM, OM, CP and energy digestibilities. height in ileum [23]. Improvement in ileal apparent digestibility co-Cost comparison: Feed cost per kg live weight gain was efficient (ADC) of CP and EE was reported [17] with significantly (P<0.01) reduced by Rs. 3.93 and Rs. 3.00 addition of avilamycin (2.5 g/kg of diet) and further in all feed additive group compared to SD and BD, improvement was observed when fortified with respectively (Table-5). Similarly, reduced cost of 8 probiotics (10 CFU/ kg) on total tract ADC for DM, production was observed in earlier studies [21] with ash, EE and ME values. The apparent digestibility of supplementation of different growth promoters such DM, OM, CP, EE, starch and energy were increased (flavomycin, avilamycin, genex and avila m/z) in (P<0.05) with supplementation of enzymes [18]. broiler diets. Feeding low calorie diet fortified with However, a previous study [15] observed no feed additives like NSP enzymes, synbiotics and improvement in apparent total tract retention of DM phytase resulted in low cost of production and better and energy as phytase or xylanase, amylase, protease returns. could influence the retention including phosphorus.

Conclusion
Tibia ash content was significantly (P<0.001) higher in supplemented groups and SD compared to From this study, it can be concluded that BD (Table 6). This might be due to synergistic effect of supplementing NSP enzyme, synbiotics and phytase in feed additives. The results are in agreement with [19] combination has exerted synergistic effect on body who reported the benefits of NSP enzymes and phytase weight gain feed conversion efficiency, improvement supplementation to broiler diets which improved bone in nutrient retentions and gut health at reduced cost of mineralization and reached to the level of positive production in broilers fed corn-soybean meal based control diet. low energy diets. Carcass characteristics: The birds receiving different 163-169. design, analyzed data and prepared the manuscript; 13. Jung S J , Houde R , Baurhoo B , Zhao X and Lee B H YRR and STVR revised manuscript. All authors read (2008) Effects of galacto-oligosaccharides and a Bifidobacteria and approved the final manuscript.
lactis-based probiotic strain on the growth performance and faecal microflora of broiler chickens. Poultry Science 87: