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Effect of Probiotic Supplementation in Broiler Birds Offered Feed Formulated with Lower Protein Densities

Shivani Katoch Sidharath Dev Rajesh Rajput
Vol 7(2), 90-102

Comparative dietary response of a combination of the different isolated bacterial strains of Lactobacilli and Streptococci isolated from leopard feces (Panthera leo) and locally procured bakery yeast Saccharomyces cerevisiae were studied as direct fed microbial (DFM) in poultry broiler birds reared on low density protein test diet under complete randomized design. For this purpose, day old broiler chicks (n=120) were divided into 3 main groups T0, T1 and T2, each group was further divided in 2 sub-groups or replicates with 20 chicks in each. Group T0 was given standard ration and served as control group. Further group T1 was given ration containing low density protein which served as negative control group and group T2 was given ration containing low density protein supplemented with direct fed microbial. The ration fed to group T1 and T2 was formulated in a way that the crude protein was 10 per cent lower than the ration fed to control group T0 but the energy content was same. Results of the experiment revealed P<0.05) higher gain in live weight and feed conversion in control group T0 fed standard diet compared to negative control group T1 and treatment group T2 fed crude protein deficient ration in both starter and finisher phase whereas treatment group T2 exhibited numerically higher gain in live weight during finisher phase compared to negative control group T1. Feed conversion ratio was (P<0.05) better in treatment group T2 fed DFM then its negative control group T1 during finisher phase which also reflected in the overall feed conversion ratio. Treatment group T2 exhibited better intestinal micro flora balance, effective colonization and higher count in the intestinal tract, lowered (P<0.05) blood cholesterol with higher (P<0.05) nitrogen retention in the digestibility studies. Thus, it was found that supplementation of isolated DFM has the potential to support growth performance of poultry broiler birds offered low density protein diet.

Keywords : Probiotic Direct-Fed-Microbial Chicken- Broiler Feed Protein Density


Economic poultry production envisages the use of optimally well balanced and efficient feeding. Interest in other growth promoters including probiotics have emerged as the result of growing concerns about prophylactic usage of antibiotic growth promoters (AGP) in poultry and other animal production systems in Europe and the US (Davis and Anderson, 2002; Mai, 2004). Digestive efficiency in chicken can be enhanced by incorporating suitable additives to their diets, like growth promoters, fed enzymes and probiotics. The use of direct fed microbial and other nontraditional feed additives has increased in response to demands for using more “natural” growth promoting substances. In this context there has been an increasing interest in the use of probiotics which have been defined as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance (Fuller, 1989) and when administered to animals in adequate amounts, benefit the host’s health. Introduction of such probiotics is believed to prevent or attenuate the growth of clinical enteric pathogens in poultry, resulting in enhanced growth and performance of the host bird. The mechanisms by which probiotics operate include spatial exclusion, micro-environmental alterations, production of antimicrobial substances and epithelial barrier integrity Chichlowski et al., 2007. Proper balance of micro organisms is an important feature of a well functioning gastro intestinal tract and a healthy intestine is dominated by lactic acid producing bacteria. This balance is disturbed by stress, disease or antibiotic treatment Lyons (1987). Lactobacilli, Streptococci and Yeast are the most commonly used groups of microbes in the production of probiotics/ DFM. The justification for the use of Lactobacilli originates from the results, that gut micro flora develops after birth, as the lactobacilli increased, other population of the flora decreased (Smith 1965). Yeast, the unicellular fungi, is also known for its fermentative ability. It produces enzymes such as amylases, proteases, lipases, celluloses as well as B-complex vitamins in the medium in which they grow. In addition yeast has a buffering effect in the digestive tract by mediating sharp drop of pH. Probiotics keep the gut at a desirable pH thus allowing the growth of beneficial bacteria which produces a natural anti biotic like substance called bacteriocins, which help to eliminate unwanted coli forms Suskovic et al. (1997). During stress or use of anti bacterial, the pH of small intestines rises which allows the growth of harmful bacteria to take a foot hold in the lining of intestine because of the deterioration of the protective mucus lining, as a result of which intestinal villi can be damaged which ultimately will inhibit nutrient absorption. Thus increasing beneficial gut micro flora through the use of Probiotic/ DFM will compete against harmful bacteria and decrease the pH of environment enhancing nutrient absorption and prevent infection (Topping 1996).

The research study was therefore carried out with the objective to study the growth performance of broiler chicken by creating a nutritional stress due to feeding a ration containing 10 per cent lower crude protein as per the NRC (1994) recommendations and stress amelioration after supplementation with direct fed microbial (DFM).

Material and Methods

The research work was carried out in the experimental poultry house of the Department of Animal Nutrition, College of Veterinary & Animal Sciences, Palampur, H.P. India. Total duration of the experiment was 42 days. For first 28 days broiler starter diet (Table2) was offered while for the remaining period broiler finisher ration (Table 2) was offered to the respective groups. For this purpose, day old vancobb broiler chicks (n=120) were divided equally into 3 main groups T0, T1, and T2 which were further divided in 2 sub groups or replicates with 20 chicks in each. Group T0was given standard ration along with culture medium and served as control group. Further group T1 was given ration containing 10 per cent lower crude protein along with culture medium and served as negative control group and group T2 was given ration containing 10 per cent lower crude protein in a culture medium supplemented with direct fed microbial as per the experimental plan (Table 1). Thus, ration fed to group T1 and T2 was formulated in a way that the crude protein was 10 per cent lower than the ration fed to control group T0 but the energy content was same. Composition of experimental ration and nutrient profile is depicted in Table 2. The difference in the physical composition of different ingredients in different treatments is due to fact that the chemical composition of the nutrients is to be kept similar in different treatments groups as per the experimental plan.

Table 1: Experimental Plan

Test Diet Treatment Source Microbial conc. / mL
T0 (Positive Control ) Standard ration Culture Medium
T(Negative control) Ration with 10 per cent lower crude protein Culture Medium
T(Treatment) Ration with 10 per cent lower crude protein Leopard feces Lactobacillus casei -6.8 x 106cells


Streptococcus fecalis 6.8 x 106cells


Saccharomyces cerevisiae 5.8 x 10cells

Standard methods as reported in (AOAC 2005) were followed for determination of proximate composition of feed and feed ingredients. The metabolizable energy (ME) contents of different test diets used under different experiments were calculated as per the equation proposed by Lodhi et al. (1976).

Isolated Direct fed Microbial (DFM)

The isolation and characterization of Lactobacillus casei and Streptococcus fecalis isolated from wild leopard (Panthera leo) feces maintained in Dhauladhar Nature park, near Palampur and Bakers’s yeast (Sacchromyces cerevisiae) procured locally was done using morphological and biochemical characteristic tests as proposed by Cowan et al. (1974).

Table 2: Ingredients and Nutrient Contents of Diets

Broiler Starter Diet (1-28d) Broiler Finisher Diet (29 to 42 d)
Ingredients (parts/100kg) Standard Diet*


Experimentally Formulated diet**

(T1, T2)

Standard Diet*


Experimentally Formulated diet**

(T1, T2)

Maize 40 40 47.3 47.3
Groundnut Cake extract (Solvent) 20 20 19 19
Whole Soybean 23 23 16 16
Fish Meal 6 6 6 6
Cotton seed Oil 2 2 3 3
Di-calcium phosphate DCP 1 1 .7 .7
Casein 3 0 3 0
Cane Molasses 5 5 5 5
Sodium Bentonite (Filler) 0 3 0 3
Premix# + + + +
Nutrients (Formulated)
Dry Matter (DM) 90.01 90.01 89.50 89.50
Crude Protein (CP) 22.92 20.62 20.79 18.49
Crude Fibre (CF) 4.60 4.60 4.35 4.35
Ether Extract (EE) 5.55 5.55 4.89 4.89
Total Ash 4.73 4.73 4.51 4.51
Nitrogen Free Extract 62.2 64.5 65.46 67.76
Calcium (Ca) 1.00 1.00 1.16 1.16
Total Phosphorus (P) 0.70 0.70 0.60 0.60
Metabolizable Energy (Kcal/Kg) 2827 2827 3135 3135

*Standard formulated diet (As per NRC-1994) ** experimentally formulated diet.

#*Premix 1) Intermix Regular: 20 g (Vit.A-82500 I.U, Vit. B2-52 mg, Vit. D1-12000 I.U, Vit. K-10 mg, Ca-166 mg, Phosphorus- 395 mg)) 2) Intermix B: 10 g (Vit B1- 4 mg, Vit. B6-8 mg, Vit. B12-40 microgram, Niacin -60 mg, Calcium Pentothenate- 40 mg) 3) DOT: 50 g (Dinitro-o-toulamide- 250mg, ethopabate, 16 mg/g) 4) E-Care Se-forte: 25 g (Vit. E 0.2g and Se 0.04mg/g) 5) Trace Minerals : 100 g (Ferric Oxide-2 gm, Copper Sulphate-2g, Ferrous Sulphate-10g, Zinc Sulphate- 0.6 g, Dicalcium Phosphate-53.65 g, Manganese Sulphate- 3 g, Magnesium Sulphate-25 g, Potassium iodide-2.5 g, Sodium thiosulphate-0.75 g, Zinc Oxide- 1 g)

Lactobacilli and Streptococci were grown in Elliker broth while Saccharomyces in Yeast fermentation broth. The techniques were standardized for the isolation, growth and preservation of different isolated microbial strains from leopard feces are as detailed below-

  1. Isolation and Characterization of Microbes from Leopard Fecal Material

Leopard fecal material was procured from “Wildlife Nature Park” Gopalpur near Palampur. A small amount of fecal material (about 2-3g) was taken and incubated in yeast fermentation broth (YFB) at 30 degree centigrade overnight. Next day, the precipitate that was deposited, was taken on a glass slide and was warmed gently on a glass flame to fix it. After fixation, staining was done with crystal violet stain for five minutes. The slides were air dried. After drying, the slides were examined under microscope in oil immersion. Mixed colonies consisting of rods, cocci, darkly staining yeast with buds and contaminants were seen. The procedure was repeated till approximately 90 per cent, contaminants were removed. Thereafter the colonies were grown on differential medium namely streptococcus selection agar (SSA) and lactobacillus selection agar (LSA).

1. For Isolation of Streptococcus, the Colonies were grown in SSA which had the Following Composition (Hi Media)

Ingredients g/litre Ingredients g/litre
Tryptone 15.00 Sodium sulphite 0.7
Soya peptone 5.00 Dextrose 5.5
Sodium chloride 4.00 Sodiumazide 0.2
L. cystine 0.2 Crystal Voilet 0.002
Agar 15.00

46.6 g of SSA was suspended in 1000mL distilled water. It was then heated till it boiled to dissolve it completely in water. The pH was adjusted to 7.0. It was then sterilized by autoclaving at 12 psi for 15 minutes. The colonies that grew on SSA were streaked repeatedly thrice on sterilized Petri plates till a pure colony of Streptococcus spp. was obtained.

2. For Isolation of Lactobacillus, the Colonies were grown in LSA (Hi Media) which had the Following Composition

Ingredients g/litre Ingredients g/litre
Tryptone 10.00 Sodium chloride 5.0
Beef extract 5.00 Dextrose 20.00
Soya peptone 4.00 L-Cysteine HCL 0.3
Yeast extract 5.0 Agar 15.00

65.3 g of LSA was suspended in 1000 mL distilled water. It was then boiled to dissolve the medium completely. Thereafter the solution was incubated at 15 psi for 15 minutes. It was then cooled to 50 degree centigrade and the following sterile solution, which was previously kept warm at 50 degree centigrade just prior to use was added.

i)100 mL of 10 per cent W/V aqueous solution of antibiotic free skim milk powder which was sterilized at 15 psi for 5 minutes and

ii) 10 mL of 2 per cent w/v TTC (Triphenly tetrazolium chloride) solution

The colonies so obtained after incubation were streaked repeatedly on sterilized Petri plates till pure colonies of Lactobacillus were obtained. The pure colonies of microbes so obtained were characterized by following the bio chemical and microscopic methods as proposed by Cowan et al., 1974 and Hucker and Conn, 1923. The isolation and characterization of the microbes was done in the specially set up laboratory for the purpose in the Department of Animal Nutrition, COVAS, CSKHPKV, Palampur in collaboration with the Department of Veterinary Public Health and Department of Veterinary Microbiology & Immunology, COVAS of the University. The microbes thus characterized were-

i) Lactobacillus casei

ii) Streptococcus fecalis

  1. Maintaining Pure Culture of Lactobacillus and Streptococcus

The pure cultures of isolated DFM from leopard fecal material i.e Lactobacillus casei andStreptococcus fecalis were grown in Elliker broth (Hi media).

The medium has the following composition-

Ingredient g/ litre Ingredient g/ litre
Tryptone 20.00 Lactose 5.0
Yeast Extract 5.00 Saccharose 5.0
Gelatin 2.5 Sodium chloride 4.0
Dextrose 5.0 Sodium acetate 1.5
Ascorbic acid 0.5

Twenty gram of Elliker broth was dissolved in 980 mL of distilled water and was warmed to dissolve the ingredients completely and the final volume was made to one litre. The pH of the medium was adjusted to 5.4. Aliquots of 100mL of the medium solution were dispersed in 250mL Erlenmeyer flasks and were sterilized by autoclaving at 15psi for 15 min and were incubated at 37 degree to check the sterilization of the medium. One per cent inoculum each of Lactobacillus casei and Streptococcus fecalis were added to the medium and incubated at 30 degree centigrade for the growth of microbes for a period of 24 hours.

Maintaining Pure Culture of Yeast

The pure cultures of Saccharomyces cerevisiae was grown in yeast fermentation broth (Hi-media) which had the following composition-

Ingredient g/ litre
Peptone 10.00
Beef Extract 5.00
Sodium chloride 5.00
Bromo cresol purple 0.04

Eighteen grams of the above medium was dissolved in 980 mL of distilled water. Glucose was added to make its final concentration to 1 per cent. pH of the medium was adjusted to 4.8. The volume was made to 1 litre. Medium was sterilized by autoclaving and incubated at 37 degree centigrade to test its complete sterilization. One per cent innoculum of pure culture of S. cerevisiae was added to the medium by taking the sterilized aliquots of 100 mL of the medium in the Erlenmeyer flasks and incubated at 30 degree centigrade. Growth of yeast was observed after 24 hours.

Standard Plate Count Method

Standard Plate count method by serial dilution was used to estimate the exact count of microbial cells as colony forming units per ml. (cfu/mL). Under aseptic conditions, one mL of nutrient broth in which the pure cultures of isolated microbes viz. lactobacillus and streptococcus were grown was taken and added to 10 mL of normal saline leading to 10 times dilution. Further, I mL of sample was taken from the 10 time diluted sample and added again to 10 mL normal saline. The procedure was repeated and serial dilutions were made eight times. 100 micro litre of sample was taken and spread uniformly to sterile Petri dishes containing LSA and SSA media in triplicate. It was then allowed to air dry in sterilized chamber and then incubated at 30 degree centigrade till growth of pure colonies was observed. The plates were then placed on a counting device, Quebec colony counter and the number of bacterial colonies was recorded. The petri plates having colony count falling between 30 and 300 were selected and multiplied by the reciprocal of the dilution factor i.e. 108 to obtain the bacterial count per mL of broth.

Management and Recording of Observations

The chicks were reared in deep litter system whose floor was covered with wood shavings at about 5 centimeter (cm) depth. Sufficient management conditions like floor space, light, temperature, ventilation, and relative humidity were provided to each of the groups. During the experimental period, they were fed ad libitum on replicate basis and provided with clean and wholesome water. Different microbial cultures (1mL diluted with 100 ml glass distilled water) as per experimental plan (Table 1) were mixed with feed quota of a particular group i.e. T1 before feeding every day and it was continued during entire period of the feeding trial. Standard management practices were followed for rearing and parameters like gain in weight (GIW), feed consumption, feed conversion ratio (FCR) and dressing percent (DP) were recorded. The data on feed intake feed left over and live weight individually were recorded using digital balance on weekly basis to calculate feed conversion ratio and gain in weight. The digestibility of various nutrients was carried out in caged battery brooders by total fecal collection method. Five birds from each experiment pen, close to the average weight of the flock were removed and placed in battery cages with wire mesh bottom and excreta collection trays. Each cage was equipped with 1 feeding and 2 water trays placed outside on the front, left and right sides of the cages. The digestibility experiment had a 3 day pre experimental adaptation period and 5 day’s collection period. During the experiment, excreta from each cage were collected and weighed using digital balance 3 times daily (i.e., with 8h intervals). Fresh droppings sample were taken for nitrogen and dry matter estimation viz., 1/100th of total excreta everyday mixed with 10mL of 10% Sulphuric acid was taken for nitrogen estimation every day in Kjeldahl digestion flasks ear marked for each sub group. Another 100 g excreta sample covered with 10mL of 10 per cent acetic acid for each sub group was kept for drying in hot air oven at 100+/- 5 degree centigrade for dry matter estimation. Dried excreta samples were ground through a 0.25 mm screen and stored at 4 degree centigrade until analyzed for other proximate principles. Remaining feed in excreta trays was carefully weighed and removed. Feathers were removed from the excreta. Feed and excreta samples drawn, were later analyzed for dry matter and CP (determined as 6.25 x Kjeldahl nitrogen) using the routine procedures (AOAC 2005). Calcium was estimated by Atomic Absorption spectrophotometer (Perkin Elmer, 1982), while phosphorus was estimated by the method proposed by Parks & Dunn (1963). All the experimental broilers were sacrificed after completion of trial to record dressing percentage.

Observation and Records

Body Weight of Chicks

The chicks of all experimental groups were weighed individually at weekly intervals. Average body weights and body weight gains for weekly interval and for entire experimental periods were then calculated.

Feed Intake

Each replicate of birds was offered measured and adequate quantity of feed both in the morning and evening of each day. Residue was collected at weekly interval. The average weekly feed intake of each group was calculated by subtracting the residue from the total feed offered and then by dividing the total feed intake in that week by the number of birds (hen days) taking into account mortality, if any.

Slaughter Records

At the end of feeding trial, two birds (1 male + 1 female) from each replicate were slaughtered to record dressing percentage. The dressing percentage was calculated as-

Dressing percentage = Carcass weight (g) × 100
Live weight (g)

Cecal Microflora

Five broilers per treatment at the age of 42 d were killed by severing the jugular vein. The carcasses were therefore opened and the entire GI tract was removed aseptically. The GI tract was first divided into different sections before separating the ceca from small intestine. Bacterial enumeration in cecal digesta per bird which was properly stored by freezing at -80ο C was done by thawing after removal from the storage bags. Cecal digesta contents were there after aseptically emptied in a new sterile bag and were immediately diluted 10 fold (i.e., 10% wt/vol) with sterile ice-cold anoxic PBS (0.1M; pH 7.0) and thereafter was homogenized using a mixer for 3 minutes. Each cecal digesta homogenate was serially diluted from 10-1 to 10-6. Duplications were subsequently placed on duplicate selective agar media for enumeration of the bacterial groups mainly Lactobacillus, Streptococci and yeast as per the methods proposed by Cowan et al. (1976). Results were expressed as log10 colony forming units per gram of cecal contents.

Blood Biochemistry

At 6th week of age, approximately 3-5 mL of blood from randomly chosen 5 birds per replicate was collected by wing vein, in a centrifuge tube containing anticoagulant heparin @ 0.1-0.2 mg/mL of blood, under aseptic conditions. The tubes were centrifuged at 5000 rpm for 15 minutes and thus plasma was separated and stored at -200 C for estimation. Cholesterol & glucose were estimated by Auto Blood Analyser using analytical kits of Bayer diagnostics.

Statistical Analysis

Data on growth performance parameters, (GIW, FCR) and nutrient apparent digestibility co-efficient were based on group basis whereas data on cecal bacterial population, dressing percent, and blood cholesterol and glucose concentration were based on individual broilers. All the recorded and calculated data were subjected to analysis of variance (ANOVA) by Duncan Multiple Range Test. Statistical significance was determined at P≤ 0.05.

Results and Discussion

The growth performance of the broilers fed different levels of protein is presented in Table 3. Average gain in live weight during starter phase was (P<0.05) highest in T(364.36) g followed by T2 (333.22) g which was numerically higher then T1 (327.84) g. Feed conversion ratio during starter phase was almost similar in T0 and T1, T2 groups and did not show any (P<0.05) difference. At the end of finisher phase, the average gain in live weight was (P<0.05) higher in T0 (944.91) g compared to T1 (798.33) g and T2 (855.76) g. DFM supplemented treatment group T2 exhibited numerically higher gain in live weight compared to T1. Feed Conversion Ratio (FCR) (Table 3) in T(2.92) was (P<0.05) better then T1 (3.31) and T2 (3.13). DFM supplemented T2 exhibited (P<0.05) better FCR compared to T1. Perusal of the results revealed that isolated DFM supplementation in treatment T2 improved weight gain during the finisher phase compared to group T1 suggesting stress-ameliorating effect of the direct fed microbial, but at the same time retarded food efficiency compared to control T0, an index of stress response. Continuing with the results, it could be made out that treatment T2 supplemented with isolated DFM was able to withstand the nutritional stress compared to its control T1 offered test diet but could not match the growth response exhibited by T0 offered standard formulated diet during finisher phase.

Table 3: Effect of Supplementing Direct-Fed Microbial (DFM) in Broiler Feed on Performance in the Starter (1 to 28 d of age) and Finisher phases (29 to 42 d of age)

Starter Phase

(1 to 28 days of age)

Finisher Phase

(29 to 42 days of age)

Overall growth performance

(1to 42 days of age)

Body Weight gram/bird Body Weight gram/bird
T DFM 28 d wt GIW FCR 42 d wt GIW FCR GIW FCR DP
T0 411.9 364.36±




1356.81 944.91±



0.06 c


29.06 a

2.60±0.02 a 61.90±1.64
T1 376.35 327.84±

4.07 b



1174.68 798.33±



0.03 a


26.18 b

2.89±0.02 c 60.34±1.15
T2 ++ 379.24 333.22±




1235.00 855.76±



0.01 b



2.76±0.01 b 63.21±1.64

Figures bearing different super- scripts are (P<0.05) different from each other. T-Treatment; D: Diet; DFM: Direct fed microbial; BW- Body weight; FBW- Final Body weight; GIW- Gain in weight; FC- Feed Consumed; FCR- Feed conversion ratio; DP: Dressing per cent

Almost similar trend in growth performance (Table 3) was obtained for different group’s viz. T0, T1 and T2 as was exhibited during the finisher phase. Birds of the group T0 exhibited (P<0.05) higher gain in live weight (1309.27) g compared to T1 (1126.17) g and T2 (1188.98) g. Overall growth results indicated that DFM supplemented treatment group T2 fed low density crude protein ration exhibited numerically higher gain in live weight compared to group T1 fed low density crude protein ration. Results obtained for feed conversion ratio (FCR) were (P<0.05) better in T0 (2.60) compared to T1 (2.89) and T2 (2.76). Further, overall results for FCR were (P<0.05) better in DFM supplemented treatment group T2 compared to T1. Experimental broilers in T2 supplemented with DFM therefore consumed less amount of feed to produce kilo gram of meat as exhibited by (P<0.05) lower value of average FCR compared to T1. The differences in mean value of dressing percent amongst T0, T1 and T2 did not show any significant difference (P<0.05) even though T2 recorded numerically 4.75 percent higher dressing yield.

A digestibility trial was conducted on 23rd day of experiment for five days. The digestibility coefficients of dry matter, crude protein, calcium and phosphorus have been presented in Table 4.Results of the digestibility trial conducted at the end of starter phase revealed non-significant (P<0.05) difference for dry matter and calcium whereas Texhibited significant (P<0.05) differences for crude protein and phosphorus compared to T1 and T2. Further, T2 exhibited (P<0.05) higher crude protein digestibility compared to Tand numerically higher calcium digestibility compared to T0 and T1.

Table 4: Digestibility, Balance of Nutrients and Blood Biochemical Estimates

Digestibility & Balance





Colonization pattern in caecal contents
T DFM DM N Ca P Cholesterol Glucose L S Y
T0 63.66±1.58 82.60±0.63 a 43.86±2.45 42.67±0.09 a 143.20±1.16 a 317.85±0.15 a 0.7±.06 b 0.2±.03 b 0.2±.03 c
T1 59.43±1.04 67.76±0.42 ab 44.22±1.43 41.39±0.02 b 95.5±2.28 b 211.85±0.26 ab 1.3±.42 b 2.5±.42 a 2.3±.55 b
T2 ++ 61.98±0.50 73.51±0.68 b 45.37±0.57 41.39±0.07 b 99.6±0.53 b 214.65±0.46 b 2.6±.21 a 3±.44 a 4.3±.49 a

Figures bearing different super scripts are (P<0.05) different from each other. T-Treatment; D: Diet; DFM: Direct fed microbial; DM: Dry matter digestibility; N: Nitrogen balance; Ca: Calcium balance; P: Phosphorus balance; Cho: Cholesterol; Glu: Glucose; L: Lactobacillus (x106cfu/mL); S: Streptococcus (x106cfu/mL); Y: Yeast (x105 cfu/mL)

The results obtained are in accordance with the findings of Nahashon et al. (1994, 1996) who reported that the addition of Lactobacillus based DFM to the diet improves N, Ca, and P retentions. There is some preliminary evidence that the administration of probiotics can affect oxalic acid excretion rates which enhances the availability of calcium in the lower gastro intestinal tract, Lieske et al., 2005. Mohan et al. (1996), Nahason et al. (1994, 1996) and Angel et al. (2005) have all reported higher nitrogen, calcium and phosphorus retention in birds supplemented with DFM. Further, Mikulec et al. (1999), Mohan et al. (1996) and El-Gawad et al. (2003) have all reported that addition of a DFM to a broiler diet result in an improvement in protein efficiency ratio as well as nitrogen retention.

The microbial colonization pattern revealed the ability of supplemented DFM to withstand and resist the acidic secretions and get established in lower parts of the intestine which are important attributes of a probiotics. Higher count of Lactobacillus, Streptococcus and Yeast count (Table 4) in the digestive tract (caecum) of experimental broilers in T2 supplemented with DFM was obtained. The highest lactobacilli count in the digestive tract (caecum) of the broilers of Treatment T2 are precisely due to the better survival and higher degree of colonization of these microbes, because after hatching of the chicks, lactobacilli are reported to attach to the mucosal lining of the crop during the course of the growth and this characteristic avoids their flushing onwards and the attached bacteria inoculate the newly ingested feed. The detached bacteria move with ingesta to the intestine which continues to influence the microbial population resulting in their dominance to compete successfully with the entero pathogens. Fuller (1986), McLean et al. (2005) while studying the effect of supplementation of Bacillus subtilis on chicken performance concluded that the micro flora in the gut appeared to have been beneficially modified (as indicated by spore counts recovered from the caecae) and thus had an effect on performance of the birds. Yub et al., 2008 reported a higher total anaerobic and Lactobacillus count at 37 days of age in all segments of gastrointestinal tract of chicks fed Lactobacillus reuteri as compared to control chicks (P<0.05). Lower value of blood cholesterol (Table 4) were obtained in T2 which exhibited significant differences (P<0.05) from T0. The results are in accordance with the findings of Chitra P et al. (2004) who reported that supplementation of probiotic showed highly significant (P<0.01) reduction of serum cholesterol level. Lowered blood cholesterol in T2 supplemented with isolated DFM are in agreement with various studies which have reported that lactobacillus can lower the total cholesterol levels and has been suggested to be related to fermentation of indigestible dietary carbohydrates. Further, products of bacterial fermentation, specifically short chain fatty acids, may inhibit cholesterol synthesis in the liver and/or cause the mobilization of blood cholesterol in the liver.

Overall trend of the results indicates a better performance by T2 supplemented with direct fed microbial (DFM) as evidenced by (P<0.05) higher feed conversion ratio and numerically higher growth compared to T1 reared on 10 per cent lower crude protein ration.


Results of the experiment thus revealed that supplementation of direct fed microbial to a protein deficient poultry broiler ration has a beneficial effect in countering the nutritional stress by maintaining a population of beneficial microflora in the gastrointestinal tract and increase in nutrient utilization through improved intestinal enzyme activities and nutrient availability.


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