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Assessing the effect of administering differentprobiotics in drinking water supplement on broilerperformance, blood biochemistry and immuneresponseSohail Hassan Khan a , Burhan Yousaf b , Asghar Ali Mian b , Abdul Rehman a & MuhammadSabir Farooq aa Department of Livestock and Dairy Development , Poultry Research Institute ,Rawalpindi, Punjab, Pakistanb Department of Poultry Science, Faculty of Veterinary & Animal Sciences , Pir Mehr AliShah Arid Agriculture University , Rawalpindi, PakistanAccepted author version posted online: 03 Oct 2011.Published online: 28 Nov 2011.

To cite this article: Sohail Hassan Khan , Burhan Yousaf , Asghar Ali Mian , Abdul Rehman & Muhammad SabirFarooq (2011) Assessing the effect of administering different probiotics in drinking water supplement on broilerperformance, blood biochemistry and immune response, Journal of Applied Animal Research, 39:4, 418-428, DOI:10.1080/09712119.2011.623783

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Assessing the effect of administering different probiotics in drinking water supplement on broiler

performance, blood biochemistry and immune response

Sohail Hassan Khana*, Burhan Yousafb, Asghar Ali Mianb, Abdul Rehmana and Muhammad Sabir Farooqa

aPoultry Research Institute, Rawalpindi, Department of Livestock and Dairy Development, Punjab, Pakistan; bDepartment ofPoultry Science, Faculty of Veterinary & Animal Sciences, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan

(Received 27 June 2011; final version received 12 September 2011)

A study was conducted to evaluate three different probiotics, using drinking water supplemented with protexin,biovet and yoghurt. The day old broiler chicks were randomly divided into 12 separate floor pens eachcomprising 25 birds and three pens (replicates) per treatment group following completely randomised design. At28 and 39 days of age body weight (BW) and feed to gain ratio (FCR) were determined. At the end of experiment,nine birds per treatment were sacrificed to evaluate carcass characteristics, abdominal fat contents and theinternal organs. Blood haemato-biochemical parameters were determined. Haemagglutination inhibition anti-body titres against Newcastle disease virus and lymphoid organs weight/body weight ratio were also determined.The BW of birds given probiotics was significantly greater than control (without probiotics) at both 28 and 39days of age. Similarly, better FCR was observed in birds those given drinking water with probiotics. There wasless mortality recorded with probiotics treatments. Differences in carcass characteristics, organs weight, meatcomposition and haematological values among all the treatments were non-significant. However, abdominal fatcontents reduced significantly in probiotics supplemented groups as compared to control and cholesterol contentswere reduced significantly supplemented groups as compared to control at both 21 and 39 days of age. Feeding ofprobiotics did positively affect the immune system within the parameters measured. It may be concluded thatperformance, blood chemistry, immunity against disease and economic efficiency in broilers could be maintainedwhen supplementing any probiotic incorporated in broiler’s drinking water.

Keywords: performance; broilers; cholesterol; meat composition; immunity

Introduction

In the modern intensive poultry production, newly

hatched chicks have little chance to contact with their

mother, thereby normal microflora is slow to colonise

in the intestine (Chegeni et al. 2011). This situation

makes chicks likely to be affected by a small number

of pathogenic bacteria due to sterile condition of

intestine, then subsequently causing food-borne dis-

ease to human beings (Alzawqari et al. 2011). A well-

accepted method to quickly introduce a commensal

microflora in hen-deprived chicks is through the

administration of probiotics. Probiotics have been

defined as live microbial feed supplement which

beneficially affects the host animal by improving its

intestinal microbial balance, have been used for the

alternative tools for helping newly-hatched chicks

colonise normal microflora as conventionally hatched

chicks do (Fuller 1989). The most widely used

probiotic strains are of the genus Lactobacillus, which

is also the dominant genus of the proximal intestine

of chickens early in life (Barnes 1979). Edens et al.

(1997) showed that in ovo and ex ovo administration

of Lactobacillus reuteri resulted in an increased villus

height, indicating that probiotics are potentially able

to enhance nutrient absorption and thereby improve

growth performance and feed efficiency. Another

species, Aspergillus oryzae and yeasts, particularly

Saccharomyces cerevisiae, have been used as probio-

tics by many workers (Montes and Pugh 1993;

Kautz and Arens 1998). Both Aspergillus spp. and

Saccharomyces belong to the Ascomycoyina subdivi-

sion and have many industrial applications, involving

brewing, distilling and baking industries (Boyd 1988).

Until recently, information on combination of mixed

bacterial-based probiotic is lacking although its

application into poultry production is on the increase.Protexin is a multi-strain probiotic containing live

microbes to establish, enhance or re-establish essen-

tial microflora in the gut. Protexin is a highly

concentrated pre-mix containing seven strains of

bacteria and two yeasts. All the micro-organisms in

the protexin are naturally occurring and have been

isolated from a wide range of feed, plant, animal, bird

and human sources. Protexin is reported to be safe,

non-toxic and residual free. There are no risks of

overdosing and protexin is compatible with all feeds,

*Corresponding author. Email: sohailhassan64@gmail.com

Journal of Applied Animal Research,

Vol. 39, No. 4, December 2011, 418�428

ISSN 0971-2119 print/ISSN 0974-1844 online

# 2011 Taylor & Francis

http://dx.doi.org/10.1080/09712119.2011.623783

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feed ingredients like vitamins and minerals and someantibiotics (International Animal Health 1999).

Biovet (as effective micro-organisms) is a probio-tic, which has recently been introduced in market. Itconsists of lactic acid bacteria, yeast, actinomycetes,fermenting fungi and antioxidants extracted fromfruits and vegetables. It is added to the drinkingwater for poultry and claims to enhance the growth,remarkable improvement in health and quality ofmeat in poultry. Mostly biovet is being used inlactating ruminant diet (Shafaqat et al. 2002; Gujjaret al. 2006). However, there is paucity of informationregarding the efficacy and beneficial effects of biovetin poultry. Without any tangible evidence fromempirically derived data, the adoption of this innova-tion in the poultry industry can not be guaranteed.

A number of dairy products are marketed ascontaining probiotic bacteria. However, the mostwidely encountered one is yogurt. Yogurt containsmainly Lactobacilli and other beneficial bacteria thathave strong positive health effects. Yogurt could aiddigestion and inhibit the development of pathogensby the improving the balance of microbes living in thedigestive tract (Metchnikott 1998). These beneficialbacteria have ability to aid in the breakdown ofproteins, carbohydrates and fats in feed and helpabsorption of necessary elements and nutrients by thehost (Shafaqat et al. 2002). They lower the pH of thegut by converting sugar to lactic acid which inhibitsthe growth of enteropathogens (Strompfova et al.2005). They have immunoregulatory actions byincreasing macrophage activity and also by enhancingthe production of immunoglobulins (Monoura et al.2008). There is a paucity of literature concerning theeffects of yoghurt as probiotic on the broiler’sperformance and need to explore the benefits of itas probiotic in the broilers chicks.

One of the proposed mechanisms responsible forbenefits of probiotic is an immunomodulatory effect(Salianeh et al. 2011). Some investigators demon-strated the potential effect of probiotic on immuno-modulation (Nayebpor et al. 2007; Apata 2008). Onthe other hand, Midilli et al. (2008) showed theineffectiveness of additive supplementation of pro-biotics on immune system. Several studies haveshown that the addition of probiotics to the diets ofbroilers leads to improved performance (Jin et al.1997, 1998). In contrast of above, Dea et al. (2006)reported that tested probiotics did not result inimprovements in broiler production efficiency overthe control group. The objective of this study was toevaluate the effects of various mixed culture probio-tics on the performance characteristics, haemato-biochemical parameters, humoral immunity andeconomic efficiency in broiler chicks.

Materials and methods

Experimental birds

A total of 300-day-old unsexed broiler chicks of aHubbard strain with initial weights of 40.7392 g wereprocured from a local hatchery where they had beenvaccinated in ovo for Marek’s disease and then rearedat Breeding and Incubation section, Poultry ResearchInstitute, Rawalpindi. The chicks were randomlydivided into 12 separate floor pens (each 10�15 feet)each comprising 25 birds and three pens (replicates)per treatment group following completely randomiseddesign. Each pen contained one tube-type feeder andone bell-type automatic water fount. Birds wereprovided with ad libitum access to feed and waterwith 23L: 1D. One 9-W fluorescent light suspended198 cm over the litter provided supplemental light ineach pen. The experimental house was thoroughlycleaned and disinfected before the arrival of birds.Care and management of the birds followed acceptedguidelines (FASS 1999). The study began on 8 June2010 and concluded 17 July 2010; thus, high summertemperatures were encountered during the study.

Experimental diets

All diets were formulated to meet or exceed NationalResearch Council (1994) recommendations for essen-tial amino acids in starter (0�28 days) and finisher(29�39 days) feeding periods. Each diet was analysedas described methods in AOAC (2000) for proximatecomposition at feed testing laboratory of PoultryResearch Institute, Rawalpindi. All analyses anddeterminations were done in triplicate. The composi-tion and calculated nutrient contents of experimentaldiets for broilers were given in Table 1.

Probiotics

Three types of probiotics were mixed in drinkingwater at the rate of protexin 1gL�1, biovet 3 mL�1

and yogurt 5mL�1. Without probiotic group isconsidered as control. In this experiment, the dosesof protexin and biovet were used as recommended bycompanies and yogurt dose was followed by findingsof Asad et al. (2005).

Protexin is a commercial probiotic which ismanufactured by Probiotics International Ltd., Eng-land and first time marketed in Pakistan by HiltonPharma (Pvt.) Ltd. An analysis of the culture mixindicated a minimum presence of 2�109 cfu g�1

(Lactobacillus plantarum, Lactobacillus bulgaricus,Lactobacillus acidophilus, Lactobacillus rhamnosus,Bifidobacterium bifidum, Streptococcus thermophilus,Enterococcus faecium, A. oryzae and Candidapinpolopesi).

Journal of Applied Animal Research 419

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Biovet is a commercial probiotic in liquid form

which is imported from Light Matrix Organics Co.,

Ltd., Switzerland and marketed in Pakistan by

Nature Farming Research & Development Founda-

tion. An analysis of the culture mix observed a

minimum presence of 1.5�109 cfu mL�1 (L. Acid-

ophilus, Bacillus subtilis, S. cerevisiae and A. oryzae).Heated milk was used for yoghurt production in

this study. Chemical composition of heated milk was

6.25 pH, 0.27% titratable acidity, 16.50% total solids,

4.26% protein and 4.80% fat. Milk used in the

production of yoghurt was preheated to 50�558Cand pasteurised at 908C for 20 min and cooled to

44918C, inoculated with mixed starter culture of L.reuteri and B. bifidum was added to the milk at a ratio

of 3%. The fermentation temperature was set at 438Cfor 8 hours. Yoghurt samples were stored at 48C.Fresh yogurt samples were prepared at feed testing

laboratory, Poultry Research Institute, Rawalpindi

and used daily during experiment period. The viable

count of the yoghurt as probiotic bacteria wasdetermined using the pour-plate method (Vinderolaet al. 2000) and the results were expressed as cfu mL�1.This analysis was done at Department of Microbiol-ogy, Quaid-e-Azam University, Islamabad, Pakistan.Selective media were used to quantify the two strains.L. reuteri was enumerated by plating the appropriatedilutions on modified Lactobacillus Selection Agar, inwhich the dextrose acting as carbon source wasreplaced by arabinose (0.3%), and the pH of themedium was adjusted to 5.0. Modified de Man,Rogosa and Sharpe (MRS) medium, with addedlithium chloride 0.3%, nalidixic acid 0.3%, neomycinsulphate 0.2% and l-cysteine hydrochloride 0.05% atpH�7, was used to enumerate B. bifidum cells. Totalpopulation of viable micro-organisms was counted onregular MRS medium (pH�5.5). All plates wereincubated anaerobically at 378C for 48 hours. Ananalysis of the culture mix showed a minimumpresence of 2.8�108 cfu mL�1 (L. reuteri and B.bifidum). The results of the present study can be usedwith confidence in evaluating possible effects ofprobiotic on performance of the broilers.

Parameter measured

Pen body weights were obtained at 7, 14, 21, 28 and39 days of experimental period. Feed consumptionwas determined for the same time periods. Birds werechecked twice daily; weight of dead birds was used toadjust for feed consumption. At the end of experi-ment, nine birds per dietary group (three per repli-cate) had their feed and water withdrawn 12 hoursprior to sacrifice and then these birds were sacrificedby severing the jugular and carotid veins after whichthey were bled. A period of 5 min was allowed toelapse between bleeding and 30 min scalding at about808C. Plucking of feathers was carried out manuallyand carefully to avoid tearing of the skin. A weight ofeach bird was taken after bleeding and plucking. Theplucked carcass were dissected according to proce-dure of Jones (1984) and eviscerated by removing theinternal organs, the head and the shank. The head,shank, abdominal fat contents and the internalorgans (i.e. gizzard, liver and heart) were separatelyweighted on a sortorious top loading chemicalbalance and likewise the eviscerated carcass. Theeviscerated carcass were then carefully cut into parts(i.e. thighs, drumsticks, breast, neck and back) andweighed separately. The respective weights of differ-ent parts of the broiler were recorded and expressedas a percentage of body live weight. For proximateanalysis, moisture, protein and fat content of rawbreasts, thighs and skin were each independentlymeasured in duplicate from each of nine broilers per

Table 1. Composition (g/kg) and calculated nutrient con-tent of basal diets.

IngredientsStarter

(1�28 days)

Finisher(29�39days)

Corn 500.00 600.00Rice broken 50.00 �Corn gluten meal (60%) 20.00 20.00Canola meal 80.00 64.00Soyabean meal (47.5%) 300.00 240.00Vegetable oil � 30.00Molasses 30.00 30.00Marble chips 5.00 5.00Dicalcium Phosphate 10.00 5.00Vitamin premixa 2.00 2.00Trace mineral mixb 1.00 1.00Choline Cl (60%)c 1.00 1.00L-Lysine HCl (98%) 1.00 2.00Total 1000.00 1000.00Calculated analysis

ME, kcal/kg 2896.60 3157.95CP, % 22.85 20.10CF, % 3.77 3.42Ash, % 7.15 6.36Available Phosphorus (%) 0.40 0.40Lysine, % 1.27 1.08Methionine, % 0.50 0.42Methionine�Cysteine, % 0.84 0.72Sodium, % 0.21 0.21Chloride, % 0.28 0.29Lino, % 1.16 3.04

aProvides per kilogram of diet: 12,000 IU of vitamin A, 4000 IU ofvitamin D3, 80 mg of vitamin E, 9 mg of menadione (vitamin K3),3 mg of thiamine, 7 mg of riboflavin, 6 mg of pyridoxine, 25 mg ofcyanocobalamin, 50 mg of nicotinic acid, 15 mg of pantothenicacid, 1.5 mg of folic acid and 150 mg of biotin.bProvides per kilogram of diet: 250 mg of Co, 1.5 mg of I, 300 mg ofSe, 50 mg of Fe, 130 mg of Mn, 20 mg of Cu and 100 mg of Zn.cProvides 1040 mg choline per kilogram of diet.

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dietary group. Samples for proximate analysis were

frozen until analysed at the Feed Testing Laboratory,

Poultry Research Institute, Rawalpindi. Moisture,

protein and fat content were measured following

AOAC (2000) methodology. To obtain total dry

matter, a unilateral sample was ground, homoge-

nised, pre-dried (658C, 12 hours) and completely

dried (1058C, 24 hours). Subsequently, Crude Protein(Micro-Kjeldahl) and ether extract (Soxhlet) analyses

were performed.On the 21st and 39th day of experiment, blood

samples were collected from the bronchial vein of

three chicks from each replicate to determine serum

cholesterol. The blood was collected in a test tube to

obtain serum. The collected blood samples were

centrifuged at 3000�g for 10 min and the serum

was decanted into aseptically treated vials and stored

at �208C for total cholesterol. Serum cholesterol was

measured using diagnostic kits (RANDOX Diagnos-

tics, Catalog No. CH 207; RANDOX Laboratories

Ltd., Ardmore, Diamond Road, Crumlin, Co., An-

trim BT29 4QY, UK) and spectrophotometer appa-

ratus. Blood samples were also analysed for packed

cell volume, haemoglobin, red blood cell, white blood

cell, lymphocytes according to the procedure of

MAFF (1984).

Antibody response against Newcastle virus

Antibody response against Newcastle disease virus

was determined by Haemagglutination Inhibition

(HI) test (Thayer and Beard 1998) at Disease

Diagnostic Laboratory, Poultry Research Institute,

Rawalpindi, Pakistan. Blood samples from each of

ten birds of each group were collected on days 7, 14,

21, 28 and 39 of post-vaccination. Serum was

separated and processed for HI test.

Lymphoid organs weight/body weight ratio

At the end of experiment, nine birds from each group

were sacrificed and lymphoid organs were weighed

separately to determine lymphoid organs weight/bodyweight ratio (Giamborne and Closser 1990).

Economic analyses

Economic analysis of live weight gain of broilerchicks was calculated by deducting net expenditurecost of chicks from the gross income of the live weightgain.

Statistical analysis

All data were determined using the SPSS version 9.5(SPSS, Cary, NC, USA) statistical analysis program.A P-value of B0.05 was considered a significantdifference among groups and the comparison ofmeans was made using Duncan’s Multiple RangeTest (Steel and Torrie 1984).

Results

Broilers performance

Supplementation of probiotics indicated significantlyincreased (PB0.05) the body weight (BW) of broilersafter 28 or 39 days of experiment (Table 2). The feedto gain ratios (FCRs) were decreased by 0.194 or0.166 units (PB0.05) for the birds with probiotictreatments during day 28 and 39 of age, respectively(Table 2) compared with control. However, BW andfeed efficiency of birds within probiotic treatmentswere similar.

The mortality percentage and the Europeanproduction efficiency factor (EPEF) are presented inTable 2. There was no significant (P�0.99) effect onmortality during the study. The mortality rate waslower for probiotic-supplemented groups (2.00, 1.75and 1.50% for protexin, biovet and yoghurt, respec-tively) than control group (3.92%) at the end ofexperiment. The mortality of the birds in the presenttrial was in the expected range and was not influencedby probiotics supplementation. The EPEF was great-er for the probiotic groups than control group.

Table 2. Effect of different probiotics on body weight (BW), feed conversion and mortality of broilers.

BW (g) Feed:gain ratio Mortality (%)

Treatments 28 days 39 days 28 days 39 days 28 days 39 days EPEFa

Control 813.09b924.32 1501.31b911.04 1.730a90.037 2.164a90.023 0.40 3.92 170Protexin 909.78a921.62 1687.71a971.44 1.582b90.036 2.020b90.075 0.00 2.00 210Biovet 923.00a948.00 1713.02a971.05 1.536b90.030 2.020b90.075 0.00 1.75 214Yoghurt 912.66a923.55 1714.82a946.69 1.597b90.062 1.998b90.024 0.00 1.50 217P-value 0.033 0.035 0.034 0.032 0.991 0.980

aEuropean production efficiency factor�liveability (%)�live weight (kg)/age at depletion (d)�FCR�100.Means with different letters differ significantly (P50.05).

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Meat yield and organ traits

The meat yield characteristics and organ traits ofdifferent groups of broilers are presented in Table 3.All these parameters showed non-significant differ-ence among treatments. However, abdominal fat wassignificantly reduced (P�0.033) by probiotic supple-mentation. Fat, moisture, and protein content ofbreast, thigh and skin were not significantly differentamong all treatments (Table 3).

Blood biochemistry

The haematological parameters were not significantlydifferent among treatments (Table 4). Effect ofdifferent probiotics on serum cholesterol of broilersat 21 and 39 days of age is presented in Table 4. Serumcholesterol concentration decreased (P�0.030) withprobiotic treatments.

Immune response

The geometric means HI titres of birds fed on dietscontaining probiotics were higher on all samplingdays than control (Table 5). The dietary supplemen-tation of probiotic resulted in increase in the antibody

titer against Newcastle disease (ND) as comparedwith that of control. The mean lymphoid organsweight/body weight ratio of control birds was sig-nificantly lower than the birds given probiotics (Table5).

Economic analysis

The economics of groups of birds supplemented withprobiotics were more encouraging as it generatedmore profit than birds on control (without probiotic).The results revealed that per bird total return on salewas US$2.02, 2.26, 2.30 and 2.30 against the totalexpenditure of US$1.69, 1.77, 1.76 and 1.75 per birdfor treatment groups control, protexin, biovet andyoghurt, respectively (Table 6). The net income perbird was US$0.33, 0.49, 0.54 and 0.55 for groupscontrol, protexin, biovet and yoghurt, respectively.Among probiotics, yogurt was more economical thanbiovet and protexin because it was locally prepared.

Discussions

It is clear from this experiment that the administra-tion of probiotics via the drinking water had

Table 3. Carcass and organ weights (expressed as percentage live weight) of broilers supplemented with different probiotics.

Treatments

Parameters Control Protexin Biovet Yoghurt P-value

Carcass (dressing%) 68.0790.10 71.9690.42 72.3090.24 72.8590.52 0.199Breast 22.5290.470 23.4590.100 23.0090.245 23.5990.350 0.185Abdominal fat 1.15a90.03 0.90b90.06 0.92b90.06 0.89b90.04 0.031Skin 4.9690.008 5.0090.007 4.9890.008 5.0890.006Back 13.0090.004 13.2190.002 13.3590.006 13.5390.002 0.645Wing 6.4890.007 6.8090.008 6.8790.009 6.8990.006 0.745Neck 2.3590.002 2.4790.003 2.4890.002 2.5390.004 0.550Head 2.6090.004 2.7390.004 2.6890.002 2.7590.001 0.530Thigh 9.7690.190 9.8990.120 9.8390.150 9.9490.200 0.795Drumstick 9.1690.003 9.3990.003 9.3490.003 9.4790.003 0.645Heart 0.4790.000 0.4890.001 0.4890.000 0.4790.001 0.152Gizzard 1.2490.002 1.3290.002 1.3890.004 1.2990.006 0.190Liver 2.2590.004 2.2390.004 2.1890.002 2.1290.001 0.189

Meat composition (%)Breast

Moisture 75.5591.3 75.1291.5 75.4091.3 75.2091.5 0.145Crude Protein 23.1991.5 22.2591.1 23.2090.9 23.2291.3 0.190Crude fat 2.0290.3 1.9390.5 1.9590.4 1.9490.5 0.175

ThighMoisture 69.0791.2 68.9291.5 69.0091.3 68.8991.5 0.173Crude protein 20.3991.5 20.2591.1 20.0891.4 20.1591.2 0.169Crude fat 11.0990.5 10.9890.5 11.0090.4 10.9490.3 0.188

SkinMoisture 42.7091.4 42.4091.4 42.5091.2 42.3091.2 0.165Crude protein 12.3591.3 12.9891.4 12.5091.5 13.0091.5 0.775Crude fat 41.0090.2 40.8290.5 40.7490.4 40.5790.3 0.180

Means with different letters differ significantly (P 5 0.05).

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beneficial effects on broiler performance. Overall, thebeneficial effects of probiotic treatments on broiler’sperformance parameters are in agreement with somenumber of other research studies using probiotics inbroilers (Kabir et al. 2004; Gil de los Santos et al.2005) compared with studies lacking positive effects(Watkins and Kratzer 1984; Priyankarage et al. 2003).However, it is difficult to directly assess differentstudies using probiotics, because the efficacy of aprobiotic application depends on many factors(Patterson and Burkholder 2003), such as speciescomposition and viability, administration level, ap-plication method (e.g. spraying, feed or water),frequency of application (e.g. once, intermittent orcontinuous), overall diet, bird age, overall farmhygiene and environmental stress factors (e.g. tem-perature and stocking density).

In the present study, the improvement of BW wasconsistent in both the growing period (0�28 days) andthe finishing period (29�39 days). This result iscontrary to the findings of Mohan et al. (1996),who observed that the beneficial effect of probiotic onchicken occurred only after the 28 days of growth,and that of Yeo and Kim (1997), who reported thataverage daily weight gain of chickens fed probioticswas significantly increased during the first 21 days ofgrowth but not during the 28�42 days of growth.Studies on the beneficial impact on poultry perfor-mance have indicated that probiotic supplementation

can have positive effects. It is clearly evident from theresult of Kabir et al. (2004) that the live weight gainswere significantly (PB0.01) higher in experimentalbirds as compared to control ones at all levels duringthe period of second, fourth, fifth and sixth weeks ofage, both in vaccinated and non-vaccinated birds. Inaddition, Torres-Rodriguez et al. (2007) reported thatadministration of the selected probiotic (FM-B11) toturkeys increased the average daily gain and marketBW, representing an economic alternative to improveturkey production.

The improvements in BW and FCR of broilers fedprobiotic supplement in the current study wereprobably due to the Lactobacillus spp., S. cerevisiae/A. oryzae and other bacterial spp. used in thesupplements. It has been suggested that to obtainthe best effects from Lactobacillus as a growthpromoting, the bacteria used must be able to surviveand later colonise the gastrointestinal tract so thattheir beneficial functions could be performed. Yo-ghurt provides an excellent carrier for probioticorganisms. It can readily utilise lactose as an energysource for growth. Thus, an important requirementfor the growth in the intestinal tract is provided bythe yoghurt. Yoghurt proteins also provide importantprotection to the probiotic bacteria during passagethrough the stomach, when compared the cellssuspended in buffered saline all cultures testedsurvived exposure to simulated gastric juice much

Table 4. Haematological and biochemical values of broiler chicks supplemented with different probiotics.

Treatments

Parameters Control Protexin Biovet Yoghurt P-value

Packed cell volume (%) 23.0090.68 23.9090.45 24.1790.75 24.4390.55 0.787Haemoglobin (g dl�1) 7.4990.33 7.7190.45 7.6390.36 8.0090.55 0.479Red blood cell (�106/mm3) 3.9090.04 4.1590.10 4.1090.08 4.2090.13 0.635White blood cell (�106/mm3) 9.9590.09 10.1090.05 10.2690.35 10.1590.44 0.175Lymphocyte (%) 33.5695.05 33.9895.30 34.3997.50 34.1596.13 0.162Cholesterol (mg dl�1) at 21 days 122.6a90.90 83.5b90.94 93.3b91.19 99.3b90.93 0.045Cholesterol (mg dl�1) at 39 days 141.0a90.89 130.3b91.10 132.5b90.95 123.0b91.20 0.033

Means with different letters differ significantly (P 5 0.05).

Table 5. Effect of probiotics on the production antibody titres against Newcastle virus and mean lymphoid organ weight/bodyweight ratio (mean9SE) of broilers.

Geometric means HI titresLymphoid organ weight/body weight ratio

(Mean9SE)

Treatments Day 7 Day 14 Day 21 Day 28 Day 39 Bursa of fabricius Thymus Spleen

Control 33.41 62.18 65.73 120.85 191.50 1.8990.003a 3.5490.005a 1.2090.004a

Protexin 52.10 96.36 143.25 211.30 310.85 2.3890.002b 4.4590.005b 2.1090.003b

Biovet 51.94 94.98 145.10 195.60 299.37 2.3590.002b 4.4190.003b 2.1590.004b

Yoghurt 52.00 92.90 142.89 197.90 298.97 2.3090.002b 4.4390.004b 2.1290.003b

P-value 0.035 0.031 0.037

Means with different letters differ significantly (P50.05).

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better in the presence of yoghurt protein (Charteris

et al. 1998).The Lactobacillus spp. in the probiotics have a

strong ability to attach to the intestinal epithelium of

chicken (Jin, Ho, Abdullah, et al. 1996), are resistant

to the bile and acidic conditions and are able to

antagonise and competitively exclude some patho-

genic bacteria in vitro (Jin, Ho, Ali, et al. 1996).

Bacterial glycolytic enzymes play an important role in

the fermentation of undigested carbohydrates and,

ultimately, in birds performance and health. The

beneficial effect of S. cerevisiae is attributed to the

fact that it is a naturally rich source of proteins,

minerals and B-complex vitamins (Davis 1976). It is

well known that yeast culture, and its cell wall extract

containing 1, 3-1, 6 d-glucan and Mannanoligosac-

charide are the important natural growth promoters

for modern livestock and poultry production (Van

Leeuwen et al. 2005). The effect of A. oryzae on

macronutrients metabolism in laying hens was ob-

served (Schneitz 2005), of which findings might be of

practical relevance. He reported that active amyloly-

tic and proteolytic enzymes residing in A. oryzae may

influence the digested nutrients.Mechanisms by which probiotics improve feed

conversion efficiency include alteration in intestinal

flora, enhancement of growth of non-pathogenic

facultative anaerobic and gram positive bacteria

forming lactic acid and hydrogen peroxide, suppres-

sion of growth of intestinal pathogens and enhance-

ment of digestion and utilisation of nutrients (Yeo

and Kim 1997). Therefore, the major outcomes from

using probiotics include improvement in growth and

improvement in feed conversion efficiency (Yeo and

Kim 1997). These results are consistent with previous

experiment of Tortuero and Fernandez (1995), who

observed improved feed conversion efficiency with

the supplementation of probiotic to the diet.Furthermore, some studies shown that the addi-

tion of E. faecium to broiler diet increased the ileal

villus height and enhanced broiler performance with

respect to weight gain and FCR (Samli et al. 2007)

and addition of a probiotic containing Lactobacilli,

Bifidobacterium thermophilum and E. faecium to

the broiler diet increased the jejunal villus height

(Chichlowski et al. 2007). Lactobacillus treatment

caused similar changes in poultry as described

previously (Dobrogosz et al. 1991). Longer villi

were found in the ileum of chicks and turkeys treated

with L. reuteri (Dunham et al. 1993) and in the ileum

of adult male layers with slight improvement in feed

efficiency after dietary addition of Bacillus subtilis

var. natto (Samanya and Yamauchi 2002). However,

above parameter regarding villus height was not

estimated in the present study, and further experi-

ments are needed to verify this effect.Broiler integrators and producers in Europe,

Africa and Asia use a ‘Production Efficiency Factor’

(PEF) to compare the live-bird performance of flocks.

This value incorporates live weight, age, liveability

and feed conversion efficiency. Under the system in

Europe, a flock with acceptable growth and liveabil-

ity parameters should attain 200�225 European PEFunits. The results of the present study indicated that

EPEF units of flocks with probiotic treatments

(210�217) were fall under acceptable growth than

control group (170).In this study, 1.5�2.0% mortality was recorded in

probiotics-supplemented groups but 3.92% mortality

was recorded in birds on control group throughout the

experiment. There were no disease or pathological

lesions obtained in the organs of slaughtered birds.

Furthermore, no medication was required for the

broilers during the experimental period. This may be

due to the fact that continuous feeding of probiotic

might have suppressed the undesirable micro-organ-

isms that lead to improved health status (build-up

resistance) and ultimately improved growth and over-

all performance. This observation could be in accor-

dance with that mention mannan Oligosaccharides

Table 6. Economic analysis of broilers on different treatments.

Treatments

Particulars Control Protexin Biovet Yoghurt

Chick cost (US $a) 0.60 0.60 0.60 0.60Feedb intake (g/bird) 3249.05 3410.22 3400.54 3426.48Cost of feed consumed (US $/bird) 0.87 0.92 0.91 0.92Others cost in US$ (probiotics, vaccine, elect., etc.) 0.22 0.25 0.25 0.23Per bird total cost (US$) 1.69 1.77 1.76 1.75Av. Live weight after 39 days (g) 1542.06 1728.45 1753.74 1755.55Return on sale at US$1.31 per kg (US$) 2.02 2.26 2.30 2.30Per bird net profit (US$) 0.33 0.49 0.54 0.55

a1US$�86.17 Pakistani rupees.bFeed price: Broiler starter�US$13.55/50 kg; Broiler Finisher�US$13.32/50 kg.

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(Extract from the cell wall of S. cerevisiae) used tocontrol pathogenic scours of all kinds in livestockcaused by Salmonella, Escherichia coli and so on(Laegreid and Bauer 2004). Fuller (2001) and Patter-son and Burkholder (2003) explained the mechanismof probiotics to pathogen inhibition by competitionfor nutrients, production of toxic condition andcompounds (volatile fatty acids, low pH and bacter-iocins), competition for binding sites on the intestinalepithelium and stimulation of the immune system.

Furthermore, the present study showed thatprobiotics supplementation did not change the broi-lers meat composition or dressing percentage andorgans weights. However, abdominal fat content wasreduced significantly. Data generated during thisstudy on meat composition and abdominal fat con-tent of broilers are in accordance with the findings ofSantoso et al. (2001). According to these workers,certain microflora present in gastrointestinal tract ofa bird impaired the absorption of cholesterol and bileacid. So there is a possibility that micro-organisms inprobiotics may cause lower absorption and deposi-tion of fat content around the abdomen and they alsosuggested probiotic could significantly decrease theactivity of acetyl-Co A carboxylase which catalysesthe rate-limiting step in fatty acid biosynthesis.

The use of probiotics in drinking water of broilershas no negative implication on the haemotologicaland health status of bird. Apparently, the haemato-logical parameters measured increased among broi-lers with probiotics over those on control treatmentbut these values were not significantly (P�0.05)different among treatments. These values were inharmony with the normal range for healthy birdsstated by Mitruka and Rawnsley (1977).

Broilers given water supplemented with probioticshad lower levels of serum cholesterol at both 28 and39 days of age than the control (without probiotic).These results lend support to the finding of theprevious experiments (Yusrizal and Chen 2003;Kannan et al. 2005). They suggested that the choles-terol-lowering effect of probiotics could be obtainedthrough retarded cholesterol synthesis and increaseddegradation of cholesterol. The hypocholesterolemiceffect by probiotic could be related to compounds inA. oryzae that is known to inhibit cholesterolbiosynthesis (Hajjaj et al. 2005). Hypocholesterolemiceffect by A. oryzae can be made by monitoring a keyenzyme, for example, 3-hydroxyl-3-methylglutaryl-coenzyme A reductase in cholesterol synthesis inpoultry (Lee et al. 2006). The hepatic synthesis ofbile acids from cholesterol is the major route ofcholesterol excretion (Wilson 1998). Certain lacticacid bacteria have the ability to produce bile salthydrolase enzyme, which deconjugates bile salts

(Klaver and Van der Meer 1993), resulting in greaterfecal excretion of bile acids (Chickai et al. 1987).Gilliland et al. (1985) hypothesised that a decrease incholesterol level could be due to the cholesterolassimilation by Lactobacillus. The probiotic supple-mentation could have enhanced the Lactobacillicount. These researchers hypothesised that someLactobacillus spp. are able to incorporate cholesterolinto the cellular membrane of the organism, thuscholesterol assimilation by Lactobacillus in turnreduces cholesterol absorption in the system, or theco-precipitation of cholesterol with conjugated bilesalt (Klaver and Van der Meer 1993).

In the present study, probiotic supplementationgreatly increased disease resistance and improvedBW. The levels of Newcastle disease vaccine (NDV)titer were higher in all the treated groups than that ofcontrol. An increase in immune titre on account ofincreased probiotic level in diet could be attributed toincrease availability of serum immunoglobulin. Pro-biotics could modulate the systemic antibody re-sponse to antigens in chickens (Huang et al. 2004).Haghighi et al. (2006) found that birds fed L.acidophilus and B. bifidum had significantly moreserum antibody to sheep red blood cells (SRBC) thanthat of unfed control. Kabir et al. (2004) evaluatedthe dynamics of probiotics on immune response ofbroilers and they reported significantly higher anti-body production (PB0.01) in experimental birds ascompared to control ones. Similarly, Khaksefidi andGhoorchi (2006) reported that the antibody titer inthe 50mgkg�1 probiotic supplemented group wassignificantly higher at 5 and 10 days of post-immu-nisation (PI) compared to control, when SRBC wasinjected at 7 and 14 days of age. Cheng et al. (2004)suggested that probiotic feeding enhanced some cell-mediated immune responses of broiler chickens bymodulating macrophage activity. It can be postulatedthat some of these effects are mediated by cytokinessecreted by immune cells stimulated with probiotic.Increased in the relative weight of bursa in broilersgiven probiotics may be attributed to increase thenumber of immune cells. Findings encountered in thisstudy is in agreement with that of Shoeib et al. (1997)who found that the bursa of Fabricious in probiotic-treated group showed an increase in the number offollicles with high plasma cell reaction in the medulla.Meanwhile, Teo and Tan (2007) observed that birdsprovided feed supplemented with B. subtilis PB6 hada significantly heavier bursa weight compared withcontrol groups. Similarly, increased in the weight ofthymus may be due to the effect of probiotic bacteriaon the functional activities of the immune systemresponses which led to increase in the number oflymphocytes in the primary lymphoid organs. The

Journal of Applied Animal Research 425

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increased in the relative weight of spleen at 39 days of

age was in agreement with the findings of Willis et al.

(2007), who found that the feeding broilers on

probiotic caused increases in the relative weights of

spleen of treatment group. In contrast, Teo and Tan

(2007) found no significant differences in the relative

weights of the spleen in broilers fed the diet contain-

ing probiotic compared with control groups. The

final diversification and affinity maturation of chick-

en b cells in spleen germinal centres was studied byArakawa et al. (1996). Following stimulation by

antigen, post-bursal b cells were able to generate

somatic variants in splenic germinal centres. The size

of these germinal centres was maximised by day 7 of

the primary response and had begun to wane by 14

days. In this study, feeding of all three probiotics

resulted in significant increase (PB0.05) in the anti-

body titer against NDV than those of control (with-

out probiotic). It was suggested that protexin, biovet

and yoghurt might be useful in treating against viral

diseases because of immunostimulating activity.Economic data clearly indicated that probiotics

supplementation was more profitable and economical

to obtain maximum gains from broiler production.

Better efficiency of feed utilisation with probiotics in

broiler drinking water was a major factor which

resulted in decreased cost of production. The results

of the present study are similar to findings of Anjum

et al. (2005), who reported that feed cost per kilogram

broiler produced was less in probiotic (protexin)

supplemented groups compared to non-supplemented

group. They concluded that protexin supplementa-

tion is more feasible and economical to obtain

maximum profitability from broiler production. Si-

milarly, Ramlah and Tan (1995) also reported that

probiotic (commercially prepared freeze dried

Lactobacillus culture) supplementation via drinking

water increases the income of broiler farmers by as

much as 11.76 cents per bird.

Conclusion

For poultry raised under commercial stress, these

products show promising effects for improving eco-

nomics of production through body weight gains,

feed: gain efficiency and improved immune system.

Based on the results, it may be concluded that

performance, blood chemistry and immunity against

disease in birds could be maintained when supple-

menting any probiotic, that is, protexin, biovet and

yoghurt incorporated in broiler’s drinking water.

However, further work is needed to clearly define

these probiotics requirement of birds.

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