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British Poultry Science

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Assessing the effect of administering probiotics in water or as a feed supplement on broiler performance and immune response
Dr M.A. Karimi Torshizi , A.R. Moghaddam , Sh. Rahimi & N. Mojgani
a a a a b

Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University, P.B. 14115-336, Tehran
b

Department of Biotechnology, Razi Vaccine and Serum Research Institute, Karaj, Iran

Version of record first published: 10 May 2010

To cite this article: Dr M.A. Karimi Torshizi, A.R. Moghaddam, Sh. Rahimi & N. Mojgani (2010): Assessing the effect of administering probiotics in water or as a feed supplement on broiler performance and immune response, British Poultry Science, 51:2, 178-184 To link to this article: http://dx.doi.org/10.1080/00071661003753756

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British Poultry Science Volume 51, Number 2 (April 2010), pp. 178184

Assessing the effect of administering probiotics in water or as a feed supplement on broiler performance and immune response
M.A. KARIMI TORSHIZI, A.R. MOGHADDAM, Sh. RAHIMI
AND

N. MOJGANI1

Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University, P.B. 14115-336, Tehran and 1Department of Biotechnology, Razi Vaccine and Serum Research Institute, Karaj, Iran

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Abstract 1. Two routes of probiotic administration in broiler farms, in water and in feed, were compared using 360 one-day-old male broiler chickens. Controls received no probiotics or antimicrobials. The water group received a probiotic preparation at a rate of 05 g/l, and the feed group received it at an inclusion rate of 1 g/kg. 2. Performance of broilers in terms body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) improved when probiotic was provided via drinking water, compared to the control and feed groups. Probiotic administration reduced plasma cholesterol and triglyceride concentrations. 3. Spleen (28 and 42 d) and bursa (42 d) relative weights were influenced by method of probiotic administration, which also improved T-cell dependent skin thickness response to phytohaemagglutinin (PHA) injection. The effect of challenge by dinitrochlorobenzene (DNCB) depended on the method of probiotic administration . 4. The method of probiotic administration can influence the performance and immune competence of birds, and administration via drinking water appears to be superior to the more conventional in-feed supplementation method.

INTRODUCTION
Probiotics are possible alternative to antibiotics as growth promoters; they are live microorganisms that contribute to the health and balance of host intestinal tract (Fuller, 1989). One of the proposed mechanisms responsible for benefits of probiotic is an immunomodulatory effect (Higgins et al., 2007b). Oral administration of lactobacilli to broiler chickens caused increased phagocytosis of enterocytes and an increase in serum IgG and IgM (Koenen et al., 2006). A large number of reports of research using probiotics on poultry have shown very variable results, from almost negative and absent effects to dramatic positive effects. Several factors could account for these discrepancies including; improper source of microorganisms, dosage levels, frequency of administration, contamination of antimicrobial chemicals, perfect environmental

and nutritional conditions (no stresses present), poor storage conditions, and poor animal welfare, as well as method of probiotic administration. Generally probiotic preparations are used on farms as soon as chicks arrive. The most common routes of administrating probiotic preparations are in feed and drinking water (Tortuero, 1973; Watkins and Kratzer, 1983) less common are in ovo injection (Cox et al., 1992; Edens et al., 1997), the use of spray (Pivnick and Nurmi, 1982; Schneitz et al., 1992, 1990; Blankenship, 1993), oral dosing (Ghadban, 2002) and more recently the vent lip method (Higgins et al., 2008). Mastbaum et al. (1997) used probiotic either via the feed or drinking water in broilers. They reported that administration of probiotics via drinking water significantly affected live weight gain and feed conversion efficiency at the end of the day 41. They also emphasised that this

Correspondence to: Dr M.A. Karimi Torshizi, Department of Poultry Science, Faculty of Agriculture, Tarbiat Modares University, P.B. 14115-336, Tehran. E-mail: karimitm@modares.ac.ir Accepted for publication 28th October 2009.

ISSN 00071668(print)/ISSN 14661799 (online)/10/0201787 2010 British Poultry Science Ltd DOI: 10.1080/00071661003753756

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beneficial effect was clearer at the end of the day 31. Arslan and Saatci (2004), administered probiotic to Japanese quail via feed and drinking water (15 g/kg or l). Regardless of route, probiotic administration improved live weight, feed conversion and feed conversion efficiency of these birds. Of the two most practical methods of probiotic administration, feed supplementation is more conventional while provision via drinking water is used to a limited extent. Providing probiotic in feed is simpler as a part of diet preparation in feed mill plants. The purpose of the present study was to compare the effects on broiler performance and immune competence of administrating probiotic in drinking water or in feed.

MATERIALS AND METHODS

Animals, diets and probiotic administration University approved methods were applied in all animal care. Three hundred and sixty 1-day-old male broiler chicks (Ross 308) were assigned at random to three experimental groups. Each consisted of 6 replications of deep litter pens (2 1 m) of 20 birds. Chicks had free access to non-medicated water and starter (121 d), and grower (2242 d) diets based on maizesoybean meal (Table 1). The temperature was set at 32 C on the first day, gradually reduced to 24 C by the third week, then maintained there to the end of experiment. The lighting pattern was 23 h L : 1 h D. Birds in the control group received no probiotic in either water or in feed. The drinking water group had probiotic (Protexin, UK, consisting of 9 different microorganisms each with similar counts, totaling up to 9 log cfu/g; the

species were Aspergillus oryzae, Lactobacillus acidophilus, L. rhamnosus, L. plantarum, L. bulgaricus, Bifidobacterium bifidum, Enterococcus faecium, Streptococcus thermophilus and Candida pintolopesii) at a concentration of 05 g/l of drinking water. Birds in the feed group received diets supplemented with the same probiotic preparation at a concentration of 1 g/kg of diet. Protexin probiotic was used because it was readily dipersable in water. The drinking water was untreated well water with following specification, pH 798, total dissolved solid 404 mg/l, electrical conductivity 620 mmohs/cm and free residual chlorine 0 mg/l. The actual total count of lactic acid bacteria in the preparation was verified by a count on MRS agar under microaerophilic conditions (Singleton, 1999). The counts for lactic acid bacteria in feed and water were not assessed. Broiler chickens maintained at moderate temperature usually consume twice as much water by weight as of feed (NRC, 1994), so in order to maintain the same amount of probiotic microorganisms received, the concentration of protexin in the-water was half of that in the feed, to approximately compensate for differences in water and feed consumption. Performance Data on performance (average daily feed and water intake, body weight gain and feed conversion ratio) were recorded weekly and are presented here on a periodic basis (121, 2242 and 142 d). Blood biochemistry Blood specimens (15 ml) were taken from the brachial vein of 6 birds per experimental group at 40 d to determine haemoglobin, cholesterol and triglyceride concentrations using commercial diagnostic kits (Pars Azmun, Iran). Immune system competence
Lymphoid organ weights

Table 1. Composition of the experimental diets (g/kg)

Ingredients Yellow maize Soybean meal Fish meal Vegetable oil Dicalcium phosphate Calcium carbonate Sodium chloride Premix1 DL-Methionine Calculated values Metabolisable energy MJ/kg Crude protein
1

Starter 5620 3300 400 378 120 100 20 50 12 1297 220

Grower 6257 2950 250 260 100 100 23 50 10 1255 200

At 28 and 42 d, 6 birds per experimental group were weighed and killed humanely. Spleens and bursae were weighed and expressed as: (organ weight/body weight) 100.
Humoral immune response to sheep red blood cell (SRBC)

Supplied per kg diet: Vit. A, 7,040 IU; Vit. D3, 2,000 IU; Vit. E, 8.8 IU; Vit. K3, 176 mg; Biotin, 012 mg; thiamine, 12 mg; Riboflavin, 32 mg; Pantothenic acid, 64 mg; Pyridoxine, 197 mg; Niacin, 28 mg; Vit. B12, 0008 mg; Choline, 320 mg; Folic acid, 038 mg; Mn, 60 mg; Fe, 60 mg; Zn, 5174 mg; Cu, 48 mg; I, 069 mg; Se, 016 mg.

SRBC was injected into the breast muscle, 01 ml of 5% (v/v, suspension in sterile PBS) of 9 chicks per treatment at 35 d. Serum specimens were taken at 40 d to determine antibody produced

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against SRBC by micro-haemagglutination test as described by Wegmann and Smithies (1966). Cellular immune response
DNCB challenge

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On day 28, 12 birds per treatment, were sensitised (Verma et al., 2004) by a single percutaneous application of 1-chloro-2,4-dinitrobenzene (DNCB-Merck). A total of 250 ml of DNCB (10 mg/ml of acetone and olive oil 4 : 1), were applied on a featherless area of the right side, whilst a similar area on the left side received the solvent without DNCB as a control. Changes in mean skin thickness before and 24 h postchallenge were assessed using digital calipers (Mitutoyo, Japan).
PHA-M induced lymphoproliferation

Phytohemagglutinin-M (Gibco, USA), T-cell mitogen was injected (100 mg dissolved in 100 ml of sterile PBS) to the right toe web of 9 birds per experimental group at 40 d. The increase in toe web thickness was measured 24 h after injection (Corrier, 1990). Statistical analysis Mean values between treatments were compared using analysis of variance followed by least significant difference test. Statistical model of ANOVA procedure of SAS (1990) used was:
Yij  Ai "ij

where Yij is the observation,  is the population mean, Ai is the administration method effect (i 13) and "ij is the residual error. Significant differences were accepted if P 005.

RESULTS AND DISCUSSION

Performance Body weight gain, feed intake, water consumption and feed conversion ratio over the starter, grower and whole period of production are presented in Table 2. Generally the probiotic via drinking water increased body weight gain of broilers throughout production periods compared to the control group (P5005). Provision of probiotic in water increased body weight gain compared to probiotic in the feed (P5005). The probiotic increased feed intake of birds during the grower phase and over the whole period compared to control (P5001). The-water group had the highest feed intake in the starter period, while the feed supplemented group had higher

feed intake than the water group during the grower and whole periods. Water intake was not influenced by probiotic provision during the first 21 d (P4005). While water intake was influenced by probiotic supplementation in a manner similar to the feed intake over 2242- and 142-d periods (P5001), it may be that this trend is a consequences of a link between water and feed intake, rather than a direct effect of probiotic administration per se. Table 2 shows water/feed ratio values. This ratio was not affected by probiotic administration (P4005). In this study the actual water/feed ratio was less than two, in spite of our initial assumption that water intake would be double food intake. Accordingly, the water group received slightly less of the probiotic preparation than the feed group; its better performance could thus not be attributed to a higher consumption than the feed group. Feed conversion ratios were affected by treatment; the lowest values were obtained in the water group (P5005). Probiotic increased daily body weight gain and feed intake of birds (Table 2). Appetite stimulation due to probiotics has been reported in laying birds by Nahanshon et al. (1993, 1996), including increased lipid, protein, and mineral retention. Feed conversion data show that the increase in body weight gain is not a simple consequence of increased feed intake, while improvement in efficiency of feed utilisation might also be involved. Improvement of feed conversion ratio was evident in the water group over control and feed groups during both grower and overall phases (P5005). Several trials have described the digestive enzyme activity of lactic acid bacteria both in vitro and in vivo (Collington et al., 1990; Jin et al., 1996a,b). Increase in total digestive enzyme activity in the treated groups (originating both from the bird (endogenous activity) and from microorganisms) could to some extent account for the improved probiotic performance in our trial. Neutralisation of enterotoxin by probiotic microorganisms and consequently better functionality of absorptive mucosal surfaces could explain, in part, the beneficial results. It seems that probiotic in water survives the demanding conditions of the upper gut, possibly due to a shorter transit time compared to solid particulate feed. The diluting ability of water might confer further benefit by reducing the negative impact of gastric acid and digestive secretions like bile and enzymes on the survivability of probiotic microorganisms in an aqueous milieu. Regardless of culture system (batch or continuous), when an inoculum of bacteria is first introduced into a growth medium, it probably requires a period to adapt to its new

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Table 2. Effect of probiotic administration in drinking water or feed on growth performance of broilers
Feed intake (g/bird/d) 121** 4534b 4714a 4536b 8754 9077 8767 066 146 030 150 079 15237b 15888a 16235a 9919b 10341a 10402a 26689b 27957ab 28637a 17721b 18517a 18701a 279 2242** 142** 121NS 2242** 142** Water intake (ml/bird/d) Water/feed (ml/g) 121NS 2242NS 142NS Feed conversion ratio 121NS 2242* 142*

Periods (d) 142** 5215c 5546a 5339b 051

Body weight gain (g/bird/d)

121*

2242*

Probiotic supplementation Control Drinking water Feed

3359b 3518a 3377b

7071b 7571a 7299b

1932 1927 1933 0014

1750 1650 1762 0012

1788 1790 1798 0010

126 125 126 0005

215ab 208b 224a 028

171ab 167b 175a 020

SEM

031

082

PROBIOTIC ADMINISTRATION VIA WATER OR FEED

NS, non-significant. abc Means with different superscripts in each column are significantly different. (*P5005, **P5001).

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surroundings the less optimal these are, the longer the period of adaptation. The length of the lag phase will also depend on the age and general health of the cells in the inoculum. During this period, there is no net increase in bacterial numbers, however the cells are metabolically active (Hogg, 2005). Commercial probiotic preparations are generally manufactured and marketed in lyophilised form, containing no free water, which is an indispensable factor required for microbial revival and proliferation. It seems that reconstitution of probiotic preparations in water could facilitate the subsequent adaptation process to a gastrointestinal milieu, by accelerating the revival process of probiotic microorganisms and consequently shorten the lag phase. These scenarios might result in a faster colonisation of probiotic microorganisms, which in turn is a pre-requisite for most of the beneficial properties of probiotics (Higgins et al., 2007a). Continuous supply of a probiotic may require pure water and might interfere with some medications and in addition may need a special device for appropriate dosing. Microorganisms are more active in hydrated conditions (in water) in comparison to the limited water content of dry feed (about 90% dry matter), which might imply more susceptibility to environmental conditions. It should be noted that some probiotic preparations are not dispersible in water, and could not be administered in the drinking water. However administration of probiotic through drinking water is not as easy as feed supplementation. Some drawbacks of this route are listed below: (1) Uneven distribution of probiotic, as sometimes chicks refuse to drink. (2) When feed is withheld from hatched chicks, application of probiotics via water is not always optimal (Ghadban, 2002). (3) Water sanitation practices. (4) Interference with water administered medications. (5) Requirement for specialised dosing devices to provide constant doses of fresh probiotic in water pipe lines. Blood biochemistry Plasma cholesterol, triglyceride and blood haemoglobin concentrations are shown in Table 3. Probiotic administration lowered plasma cholesterol and triglyceride concentrations compared to control group (P5001), while blood haemoglobin was not influenced by probiotic and its different administration routes (P4005). Cholesterol and triglyceride concentrations in plasma were significantly reduced by probiotic application (Table 3). The route of administration was not significant, although the water group showed lower plasma cholesterol in comparison to the feed group. The lowering of

Table 3. Effect of probiotic administration in drinking water or feed on plasma cholesterol and blood haemoglobin
Probiotic supplementation Plasma Plasma HaemoglobinNS cholesterol* triglyceride* mg/dl 15595a 13025b 13891ab 422 12589a 9086b 9048b 635 752 727 734 008

Control Drinking_water Feed SEM

NS, non-significant. ab Means with different superscripts in each column are significantly different. (*P5001).

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cholesterol by some microorganisms, is well documented, though the mechanism remains to be clarified (Klaver and van der Meer, 1993; Jin et al., 1998b, Mohan et al., 1996). There is uncertainty regarding probiotics competiting for some nutrients, especially folic acid, within the host animal. Table 3 shows that in this study the probiotic did not influence haemoglobin concentrations, which suggests any competitive interaction was absent. Immune system competence
Lymphoid organ weights

Spleen (28 and 42 d) and bursa (42 d) relative weights were influenced by probiotic administration. Spleen (P5001 at 28 d, P5005 at 42 d) and bursa (P5001 at 42 d) relative weights were highest in the water group, with the lowest values in the control group. Gore and Qureshi (1997) used relative lymphoid organ weight (bursa of Fabricius and spleen) as criteria for immune response competence, with heavier weights indicating higher immune competence. Our findings showed lymphoid organ weights were generally heavier when probiotic was administered (Table 4) and were greater in the water than in the feed group. Treatment had a clear effect at 28 d but at 42 d there was no difference between water and feed groups. In contrast, Jin et al. (1998a), who administered probiotics in the feed, found no significant effect of single strain or mix culture of 12 lactobacilli on relative weights of spleen and bursa of broilers. Humoral and cellular immune response Antibody titre against SRBC was not affected by administration route (P4005), whereas skin response to DNCB challenge or PHA injection was influenced by it. The greatest skin response to DNCB (P5005) and PHA (P5001) was in the water group.

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Table 4. Effect of probiotic administration in drinking water or feed on the relative weights of lymphoid organs, haemagglutinin titres against SRBC (HA titre), cell mediated immunity as assessed by contact sensitivity to DNCB and PHA-M injection
Sampling time Relative weight (g/100 g body weight) Spleen 28 d** Probiotic supplementation Control Drinking water Feed SEM 42 d* 28 dNS Bursa 42 d** HA titre (Log 2) 40 dNS Increase in skin thickness (%) to DNCB 28 d* PHA 24 h**

0095b 0138a 0088b 0008

0081b 0119a 0119a 0007

0185 0188 0172 0004

0130b 0204a 0191a 0012

233 250 233 011

160b 226a 182b 011

064c 116a 089b 008

NS, non-significant. abc Means with different superscripts in each column are significantly different. (*P5005, **P5001).

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The immune modulation property of probiotics has been well addressed (Koenen et al., 2004; Haghighi et al., 2005; Higgins et al., 2007b). Probiotic administration through drinking water slightly improved antibody production against SRBC in comparison to control and probiotic fed groups (P4005). Haghighi et al. (2006), reported enhancement of serum and intestinal natural antibodies to several foreign antigens. Probiotic administration via drinking water significantly improved cellular immune response to DNCB and PHA, while probiotic provision in the feed improved cellular immune response to PHA injection compared to control (P5001). However, enhanced immune response to DNCB challenge was not significant in probiotic fed group in comparison to control (P4005). DNCB challenge has been successfully applied (Swamy et al., 2004; Verma et al., 2004) to test cellular immune response in broilers fed mycotoxincontaminated feed. To our knowledge, the current study is the first report of DNCB test used in probiotic immune-stimulating assays. Probiotic enhances the immune competence of broilers by macrophage activation, increase of systemic and local antibody production (Ahmad, 2006). In conclusion, our results showed that probiotic administration benefited broiler performance, some blood biochemical values and immune modulation. The method of administration affected probiotic efficiency, with drinking water as the method of choice because of its greater efficiency.

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ACKNOWLEDGEMENTS
Funding provided by the Research Affairs of Tarbiat Modares University, Iran is acknowledged. The authors thank Afshin Khakpour for improving the manuscript.

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