Characteristics of effective probiotics

Just as not all strains of bacteria are the same, not all probiotics are the same. The effectiveness of a probiotic supplement depends upon what it contains. A good probiotic should have the following characteristics:

• • The culture should be acid and bile resistant and should contain a minimum of 30,109 CFU (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003; Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008).

• • It should be strain specific. The culture should possess high survival ability and multiply fast in the conditions within the poultry gut (Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008).

• • The culture should not have any side effects. It should be neither pathogenic nor toxic to the host (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003; Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008).

• • The culture should have a strong adhesive capability with the digestive tract of the poultry (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003).

• • Be durable enough to withstand the duress of commercial manufacturing, processing and distribution (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003).

• • The culture should have the ability to reduce pathogenic microorganisms (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003; Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008).

• • It should be able to modulate immune response (Patterson and Burkholder, Reference PATTERSON and BURKHOLDER2003).

Modes of action of probiotics

The beneficial effects of probiotics are mediated by their mechanism of action through which they inhibit the growth and proliferation of pathogenic bacteria. The most common manner of inhibition is by lowering the pH of the gut. During in vitro studies it was found that primary metabolites, such as organic acids and hydrogen peroxide, are involved in the suppression of bacterial cultures (Fuller, Reference FULLER1989). Later volatile fatty acids (VFAs) were found to be equally effective in the suppression of pathogenic gut flora (Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007). Probiotics produce VFAs and organic acids as part of their natural breakdown and metabolism of nutrients in the gut digesta. These organic acids lower the pH below that essential for the survival of pathogenic bacteria such as E. coli and Salmonella spp.. It is now well established that the observed beneficial effects of probiotics is accomplished via lowering the pH through the production of VFAs which inhibit the growth of harmful bacteria (Fuller, Reference FULLER1989; Pascual et al., Reference PASCUAL, HUGAS, BADIOLA, MONFORT and GARRIGA1999; Yoruk et al., Reference YORUK, GUL, HAYIRLI and MACIT2004; O'Dea et al., Reference O'DEA, FASENKO, ALLISON, KORVER, TANNOCK and GUAN2006; Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007; Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008). Another mechanism is through the competition for adhesion sites on the intestinal epithelium, thus preventing colonies of pathogenic bacteria forming (Guillot, Reference GUILLOT2003; O'Dea et al., Reference O'DEA, FASENKO, ALLISON, KORVER, TANNOCK and GUAN2006; Revolledo et al., Reference REVOLLEDO, FERREIRA and MEAD2006; Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007; Choudhari et al., Reference CHOUDHARI, SHINDE and RAMTEKE2008). This ‘competitive exclusion’ of harmful bacteria is achieved through colonisation of favourable sites of adhesion such as the intestinal villus and colonic crypts, or excretion of the mucins (MUC2 and MUC3) from goblet cells which inhibits the adherence of enteropathogenic bacteria (Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007). Competitive exclusion via probiotics depends upon the ability of the strain to adhere to the gut surface which is a host specific phenomenon and varies from strain to strain within the same species (Fuller, Reference FULLER1989). Lactic acid bacteria are well known to colonise the caecal wall in the chicken and their competitive exclusion effect has been explained (Starvic, Reference STARVIC1987; Fuller, Reference FULLER1989; Yoruk et al., Reference YORUK, GUL, HAYIRLI and MACIT2004). This stresses the point that a strain adhering well to the gut should be chosen while selecting a probiotic.

Another important mechanism involved in producing beneficial impacts on the host's body is the stimulation of the immune system. An accumulated body of evidence has shown that the protective effect of probiotics is associated with elevated humoral and cellular immune responses, which is achieved through increased production of T lymphocytes, CD⁺ cells and antibody secreting cells, expression of pro- and anti-inflammatory cytokines, interleukins, IFN-gamma, natural killer cells, antibody production, respiratory burst in macrophages and delayed type hypersensitivity reactions (Panda et al., Reference PANDA, REDDY, RAMA and PRAHARAJ2003; Oyetayo and Oyetayo, Reference OYETAYO and OYETAYO2005; Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007; Musa et al., Reference MUSA, WU, ZHU, SERI and ZHU2009; Ohashi and Ushida, Reference OHASHI and USHIDA2009). Another mode of action of probiotics is lowering the activities of the intestinal and faecal β-glucosidase and β-glucuronidase bacterial enzymes. These enzymes are involved in the formation of toxins in the body. The lactobacillus culture may reduce β-glucosidase and β-glucuronidase activities by attaching themselves along the chicken intestine, thus preventing colonisation of the bacteria with toxicant-promoting enzymes (Jin et al., Reference JIN, HO, ABDULLAH and JALALUDIN2000). Additionally, lyzozyme produced by Bifadobacteria, has been reported to alter the pathogenic activities of bacteria, reduce antibiotic-induced side-effects, inhibits mammary and liver tumours and in conjunction with oligofructose decrease 1, 2-dimethylhydrazine induced carcinogenesis (Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007).

Competition for nutrients in the gut, especially carbohydrate, is well recognised (Fuller, Reference FULLER1989; Choudhri et al., 2008). Probiotic organisms compete with pathogens for nutrients thus preventing them from acquiring energy for growth and function in the gut environment (Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007). In vitro studies have demonstrated competition for carbon sources between the gut flora and Shigella flexneri (Fuller, Reference FULLER1989). Inhibition of bacterial toxins by probiotics has also been reported (Brandao et al., Reference BRANDAO, CASTRO, BAMBIRRA, AMARAL, FIETTO and TROPIA1998; Musa et al., Reference MUSA, WU, ZHU, SERI and ZHU2009), which involve several mechanisms. Firstly, probiotics produce 54-kDa protease which digests the toxin and its receptor, through which the toxin attaches to the enterocyte (Pouthoulakis et al., Reference POUTHOULAKIS, KELLY, JOSHI, GAO, O'KEANE, CASTIGLIOULO and LAMONT1993; Brandao et al., Reference BRANDAO, CASTRO, BAMBIRRA, AMARAL, FIETTO and TROPIA1998). Secondly, probiotic bacteria reduce the formation of cylic AMP (cAMP) of the intestine. E. coli and cholera toxins catalyse the activation of adenyle cyclase causing a rise in cAMP that triggers active secretion of chloride and bicarbonate in crypt cells and inhibits water absorption in the villus resulting in diarrhoea. S. boulardii was demonstrated to produce a 120-kDa protein, which reduces the formation of cAMP by intestinal cells to which E. coli thermo labile toxins has been added (Czerucka et al., Reference CZERUCKA, ROUX and RAMPAL1994). Thirdly, the specific toxin may adhere to the probiotic surface. If specific receptors of the toxin are similar to the surface receptor of S. boulardii membrane, there is a likelihood that the toxin may bind to the probiotic bacteria (Brandao et al., Reference BRANDAO, CASTRO, BAMBIRRA, AMARAL, FIETTO and TROPIA1998). It has also been demonstrated that probiotics produce antimicrobial substances which prevent the pathogenic bacteria from localising in the animal gut (Fuller, Reference FULLER1989; Vandenbergh, Reference VANDENBERGH1993). This class of small antimicrobial molecules, referred to as bacteriocins, defensins and cathelicidines, act to combat the pathogenic bacteria or impede their colonisation. These are protein or protein complexes which have an antagonistic effect against the pathogenic bacteria. The polyamine derived piperidine, yielded by the intestinal microflora as a result of amino acid degradation, has been shown to inhibit the binding of Salmonella and Shigella to the intestinal epithelial cells (Chichlowski et al., Reference CHICHLOWSKI, CROOM, MCBRIDE, HAVENSTEIN and KOCI2007).

Body growth and feed conversion ratio (FCR)

Panda et al. (Reference PANDA, REDDY, RAMA and PRAHARAJ2003) reported that the inclusion of L. sporogens (100 mg/kg feed) resulted in an increased body weight and improved FCR in commercial broilers. In another study, the addition of probiotic (L. acidofillus and S. faecium) to broiler feed significantly improved the growth rate (Mohan et al., Reference MOHAN, KADIRVEL, NATARAJAN and BHASKARAN1996; Choudhary et al., 2008). The live yeast culture of S. cerevisiae along with L. acidophillus and S. faecium were supplemented in broiler feed (1 kg/ton) and the results showed an improved weight gain and FCR (Choudhary et al., 2008). In laying hens, Lactobacilli spp. supplementation resulted in improvement in feed efficiency and egg production (Mohan et al., Reference MOHAN, KADIRVEL, NATARAJAN and BHASKARAN1996). Balevi et al. (Reference BALEVI, UCAN, COSKUN, KURTOGLU and CETINGUL2001) found that supplementation of the diet with a commercial probiotic (Protexin™) at 500 g/tonne resulted in improved feed intake, body weight gain and FCR. Yoruk et al. (Reference YORUK, GUL, HAYIRLI and MACIT2004) reported that FCR improved linearly with increasing level (0.1% and 0.2%) of probiotics (Lactobacilli spp. + Enterococcus faecium + Bifadobacterium bifidum + Aspergillus oryza) in hens during the late laying period. Eckert et al. (Reference ECKERT, LEE, D., STEVENS, ANDERSON, ANDERSON, BELTRAN, SCHATZMAYR, MOHN and CALDWELL2010) reported that body weight and FCR were improved in response to Lactobacillus- based probiotics. Similarly, Zhu et al. (Reference ZHU, ZHANG, WU and ZHANG2009) reported that Lactobacillus salivarius improved body weight gain and FCR.

O'Dea et al. (Reference O'DEA, FASENKO, ALLISON, KORVER, TANNOCK and GUAN2006) examined probiotic mixtures (Lactobacillus acidophilus, Lactobacillus bifidus, and Streptococcus faecalis) using different regimes and concluded that weight gain improved significantly in broilers produced by 43 and 57 week old breeder flocks fed the supplemented diet. Accumulated evidence suggests that inactivated probiotics could have similar beneficial effects to those of live probiotics. Huang et al. (Reference HUANG, CHOI, HOUDE, LEE, LEE and ZHAO2004) reported that inactivated probiotics, after disruption with a high pressure homogeniser, have beneficial effects on the productivity of broiler chicks when used at a certain concentration. They also found that body weight gain was improved with disrupted, cobalt-enriched lactic acid bacteria (L. acidophilus and L. casei) and fungal mycelium (S. acidophilum), when sprayed onto a mash basal diet. Zhou et al. (Reference ZHOU, WANG and LI2010) found that Bacillus coagulans ZJU0616, improved growth performance, FCR, breast chemical composition and meat quality of Guangxi Yellow chickens. Hassanein and Soliman (Reference HASSANEIN and SOLIMAN2010) found that supplementing with a live yeast culture of Saccharomyces cerevisiae at the level of 0.4% and 0.8% improved FCR in White Leghorn birds. Panda et al. (Reference PANDA, RAO, RAJU and SHARMA2008) reported that dietary preparation of L. sporogenes at 100 mg (6 × 108 spore) per kg of diet significantly enhanced feed efficiency in White Leghorn breeders, which was ascribed to the beneficial effects of probiotic feeding on digestion and utilisation of nutrients. In the same study, no positive effect of this probiotic was recorded on body weight gain and feed intake. Zhu et al. (Reference ZHU, ZHANG, WU and ZHANG2009) described that the degree of a probiotics effect depends upon species, bacterial strain, application method, bird's age, overall hygiene conditions on farm and environmental factors.

Egg production and egg quality

Probiotics increase egg production, improve egg quality and decrease egg contamination (Haddadin et al., Reference HADDADIN, ABDULRAHIM, HASHLAMOUN and ROBINSON1996; Kurtoglu et al., Reference KURTOGLU, KURTOGLU, SEKER, SKUN, BALEVI and POLAT2004; Van Immerseel et al., Reference VAN IMMERSEEL, RUSSELL, FLYTHE, GANTOIS, TIMBERMONT, PASMANS, HAESEBROUCK and DUCATELLE2006). Further, it has been reported that probiotics increase egg shell weight, shell thickness and serum calcium in layers (Panda et al., Reference PANDA, REDDY, RAMA and PRAHARAJ2003). Balevi et al. (Reference BALEVI, UCAN, COSKUN, KURTOGLU and CETINGUL2001) found that commercial probiotic (Protexin™) supplemented diets resulted in a decreased broken egg ratio in layers. Yoruk et al. (Reference YORUK, GUL, HAYIRLI and MACIT2004) found that egg production increased linearly with increasing levels of probiotics (Lactobacilli spp. + Enterococcus faecium + Bifadobacterium bifidum + Aspergillus oryza) during the late laying period in layers. Panda et al. (Reference PANDA, REDDY, RAMA and PRAHARAJ2003) reported no improvement in FCR, however, increased egg production, shell weight, shell thickness and serum calcium were recorded by feeding a Lactobacillus based probiotic at the rate of 100 mg/kg and 200 mg/kg of feed, although there was no significant difference between the two treatments. In the same experiment, the egg shell and serum calcium improved significantly. Panda et al. (Reference PANDA, RAO, RAJU and SHARMA2008) reported that dietary preparation of L. sporogenes at 100 mg (6 × 108 spore) per kg of diet significantly increased egg production, egg shell breaking strength, shell weight and shell thickness in laying hens. However, egg weight, specific gravity and Haugh unit was not influenced. Several studies with layers indicated that supplementation of probiotics had no positive effect on egg production (Balevi et al., Reference BALEVI, UCAN, COSKUN, KURTOGLU and CETINGUL2001; Nahashon et al., Reference NAHASHON, NAKAUE and MIROSH1994; Panda et al., Reference PANDA, RAO, RAJU and SHARMA2008). Variations in the effect of probiotics obtained from various studies have been attributed to the difference in strains, form of bacteria, their concentration in the diet and viability in the gastrointestinal tract (Panda et al., Reference PANDA, RAO, RAJU and SHARMA2008).

Nahashon et al. (Reference NAHASHON, NAKAUE and MIROSH1994) recorded a greater shell thickness following supplementation with Lactobacillus-based probiotics in Single Comb White Leghorn layers. The beneficial effects on egg shell have been attributed to the favourable environment in the intestinal tract resulting from probiotic supplementation which may absorb more calcium from the gut, leading to thicker egg shells. However, a probiotic (Lactobacillus acidophilus, Lactobacillus bifidus and Streptococcus faecalis) showed no effect on chick quality in broilers produced from breeder flocks at various ages (O'Dea et al., Reference O'DEA, FASENKO, ALLISON, KORVER, TANNOCK and GUAN2006). Hassanein and Soliman (Reference HASSANEIN and SOLIMAN2010) reported that live yeast culture of Saccharomyces cerevisiae at the level of 0.4% and 0.8% resulted in a significant increase in egg production, egg albumin, egg yolk and egg shell thickness in White Leghorn layers.

Reducing food borne bacteria

Intensive genetic selection in broilers and layers in recent years for high performance traits has resulted in an increased susceptibility to infectious diseases. Poultry meat has been associated with the transmission of enteric pathogens, including Salmonella and Campylobacter spp (Cox and Pavic, Reference COX and PAVIC2009). Callaway et al. (Reference CALLAWAY, EDRINGTON, ANDERSON, BYRD and NISBET2008) stated that the ‘link between human salmonella and host animals is most clear in poultry’ and that raw eggs and undercooked poultry are considered to be hazardous. Eggs have been implicated as vehicles in numerous outbreaks of salmonella; in particular, eggs are major vehicle of transmission of strains of Salmonella enteritidis (Cox and Pavic, Reference COX and PAVIC2009). Probiotics have been extensively used to control pathogenic Salmonella in chickens to reduce mortality. Salmonella is one of the most important food borne zoonotic diseases around the world (Pascual et al., Reference PASCUAL, HUGAS, BADIOLA, MONFORT and GARRIGA1999). Salmonella spp. contamination of poultry products primarily originates from the GIT of poultry, specifically the caeca, where there is high microbial activity. To produce salmonella-free meat and eggs, recent research has focused on reducing salmonella infection through competitive exclusion. The specific strains of Lactobacillus spp. adhere to the wall of the intestines of the host and competitively eject the salmonella from the gut. Hassanein and Soliman (Reference HASSANEIN and SOLIMAN2010) found that live yeast culture of Saccharomyces cerevisiae at the level of 0.4% and 0.8% decreased the intestinal load of Escherichia coli, Klebsiella sp., Staphylococcus sp., Micrococcus sp., Campylobacter sp., and Clostridium perfringens in layers.

When poultry meat and eggs were recognised as a vehicle for human salmonella, the application of probiotics as a tool for preventing this disease was actively explored. Cox and Pavic, (Reference COX and PAVIC2009) reported that increased numbers of Lactobacillus and Bifadobacterium spp. correlated with reduced Salmonella spp. prevalence. Starvic (Reference STARVIC1987) treated newly hatched chicks with different strains of bacteria belonging to Lactobacillus, Bacteroides, Escherichia and Streptococcus spp, and observed an increased inhibition of Salmonella spp. colonisation. Pascual et al. (Reference PASCUAL, HUGAS, BADIOLA, MONFORT and GARRIGA1999) reported that a single strain of lactobacillus salivarius was capable alone of eliminating Salmonella enteritidis from the gut of one day old chicks. The immunological properties of probiotics have been extensively studied, demonstrating that certain Lactobacilli augment systemic and mucosal immunity against enteropathogens, leading to the production of secretory IgA (Revolledo et al., Reference REVOLLEDO, FERREIRA and MEAD2006). The beneficial effects of probiotics, however, depend upon the health of the birds, which determine the extent of colonisation by enteropathogens (Pascual et al., Reference PASCUAL, HUGAS, BADIOLA, MONFORT and GARRIGA1999).

Probiotics have been applied for the prevention of Campylobacter jejuni infection in poultry. C. jejuni is considered to be one of the major causes of food borne bacteria. Researchers have explored the ability of Lactobacillus spp. in producing anti-Campylobacter jejuni compounds to reduce infection. Doyle and Schoeni (Reference DOYLE and SCHOENI1986) reported on the selection of bacteria from chickens with the ability to produce anti-C. jejuni metabolites. They concluded that chicks treated with probiotics had an average protection of 64% against C. jejuni when compared to the control group. In the same study, the effect of probiotic supplementation with lactose, mannose and fructoligosaccharides on the extent of inhibition of C. jejuni was explored. These compounds were found to enhance the effectiveness of probiotics. Recently, Stern et al. (Reference STERN, ERUSLANOV‌, POKHILENKO‌, KOVALEV‌, VOLODINA‌, PERELYGIN‌, MITSEVICH‌, MITSEVICH, BORZENKOV‌, LEVCHUK‌, SVETOCH‌, STEPANSHIN‌ and SVETOCH2008) fed 250 mg of purified bacteriocins per kg feed to broiler chicks and found that bacteriocins (obtained from Lactobacillus salivarius and Paenibacillus polymyxa) substantially reduced C. jenuni colonisation in live birds. Cox and Pavic (Reference COX and PAVIC2009) reported that competitive exclusion through probiotics may provide the best tool to exclude Salmonella spp., however, under commercial conditions, degree of exclusion of Salmonella spp. has been highly variable as the efficacy of competitive exclusion requires salmonella-free chicks, good biosecurity and low stress levels during the first few days of treatment, which may not be practical or possible. Recently, Santini et al. (Reference SANTINI, BAFFONI, GAGGIA, GRANATA, GASBARRI, DI GIOIA and BIAVATI B.2010) suggested that Bifadobacterium longum PCB 133, possesses high probiotic properties and marked anti-campylobacter activities both in vivo and in vitro, and is an excellent candidate as a feed additive for poultry for the reduction of food-borne campylobacteria in humans. Higgins et al. (Reference HIGGINS, ERF, HIGGINS, HENDERSON, WOLFENDEN, GAONA-RAMIREZ and M. HARGIS2007) suggested that macrophages are directly or indirectly involved in the diminution of salmonella colonisation caused by the administration of probiotics.

Stimulation of immunity

The gut and its resident microflora play a significant role in shaping the immune system (Diarra et al., Reference DIARRA, KWARI, GIRGIRI, SALEH and IGWEBUIKE2011). Commensal bacteria in the intestine have intimate contact with the cells of the gut-associated immune system (Haghighi et al., Reference HAGHIGHI, GONG, GYLES, HAYES, SANEI, PARVIZI, GISAVI, CHAMBERS and SHARIF2005). The oral administration of probiotics mainly promotes the activities of splenic NK cells and stimulates phagocytic activity (Matsuzaki et al., Reference MATSUZAKI, YAMAZAKI, HASHIMOTO and YOKOKURA1998). An oral dose with native gut flora from salmonella-free adult birds may protect chickens from salmonella infections by improving their immune status (Starvic, Reference STARVIC1987). Panda et al. (Reference PANDA, RAO, RAJU and SHARMA2008) reported that a dietary preparation of L. sporogenes at 100mg (6 × 10⁸ spore) per kg of diet significantly increased humoral response in laying hens. The increase was ascribed to the increased activities of antibody producing cells, which might have been stimulated by the probiotic organism (Panda et al., Reference PANDA, REDDY, RAMA and PRAHARAJ2003).

Panda et al. (Reference PANDA, REDDY, RAMA and PRAHARAJ2003) recorded significantly higher antibody titres and coetaneous basophilic hypersensitivity (CBH) in layers supplemented with probiotics (100 mg/kg of feed). Supplementation of probiotics in layers increases cellularity of Payer's patches in the ileum, an indication of stimulation of the mucosal immune system, which secrete immunoglobulin (IgA) in response to antigenic stimuli (Nahashon et al., Reference NAHASHON, NAKAUE and MIROSH1994). Probiotic-treated birds have significantly more serum antibody (IgM) against SRBC (sheep red blood cells) than birds that are not treated with probiotics (Haghighi et al., Reference HAGHIGHI, GONG, GYLES, HAYES, SANEI, PARVIZI, GISAVI, CHAMBERS and SHARIF2005). Antibody titres were augmented in Newcastle, bronchitis and influenza infections by administering probiotics, either singly or in combinations (Starvic, Reference STARVIC1987). The higher antibody titres seen in birds supplemented with probiotics has been attributed to the beneficial effect of microflora on the gut to maintain a sound balance of immunopotent cells (Panda et al., Reference PANDA, REDDY, RAMA and PRAHARAJ2003). Accumulated evidence has shown that inactivated L. acidophilus and L. casei enhance IgA titres in the serum of broiler chicks (Huang et al., Reference HUANG, CHOI, HOUDE, LEE, LEE and ZHAO2004). Li et al. (Reference LI, ZHAO X.J. and WANG2009) investigated the effect of probiotics (Lactobacillus and Bacillus cereus) on the immune status of broiler chicks and found that, compared to control birds, Newcastle disease antibody titres, T-lymphocyte percentage and immune organ relative weights increased significantly. Higgins et al. (Reference HIGGINS, ERF, HIGGINS, HENDERSON, WOLFENDEN, GAONA-RAMIREZ and M. HARGIS2007) reported that Lactobacillus-based probiotic cultures improved the number of macrophages in the caecum as well as increasing the phagocytic activities against Salmonella enteritidis, suggesting that probiotics have the ability to modulate the innate immunity of broilers.

Improve bone strength

Skeletal disorders are well established in broilers due to their fast growth. The bones provide structural support as well as a reservoir for minerals during metabolism (Mutus et al., Reference MUTUS, KOBAGLI, ALP, ACAR, EREN and GEZEN2006). Various studies suggest that probiotics improve bone strength and development. Mutus et al. (Reference MUTUS, KOBAGLI, ALP, ACAR, EREN and GEZEN2006) showed that the morphometric properties of the tibial bone in broilers were affected by supplementation of probiotics in the diet. Plavnik and Scott (Reference PLAVNIK and SCOTT1980) reported that a diet supplemented with 2.5 or 5.0% brewer's yeast probiotic reduced leg weakness in broilers. A diet supplemented with probiotics resulted in better Ca and P retention and higher than normal mineral deposits in the skeleton (Nahashon et al., Reference NAHASHON, NAKAUE and MIROSH1994). Mutus et al. (Reference MUTUS, KOBAGLI, ALP, ACAR, EREN and GEZEN2006) conducted a trial on broilers fed probiotics for six weeks. They found that dietary probiotic supplementation improved the thickness of the medial and lateral wall of the tibia, percentage ash, tibiotarasl index and P content, which are indicators of bone strength. In the same experiment, probiotic supplementation slightly improved the tibia yield stress and modulus of elasticity of birds, which are parameters of the rigidity of the bones.

Decrease parasitic load

Lee et al. (Reference LEE, LILLEHOJ, DALLOUL, PARK, HONG and LIN2007a) found that in-feed probiotics can be effective against Eimeria tenella and E. acervulina infections. They found that feeding 0.1% probiotics orally reduced oocyte shedding in broilers infected with 5,000 CFU E. acervulina, resulting in improved weight gain. In the same experiment, eimeria-specific antibody levels were significantly higher compared to the unsupplemented control birds. These results suggest that probiotics effectively enhance the resistance of birds and protect them against the negative growth effects associated with coccidiosis. In another trial, Lee et al. (Reference LEE, LILLEHOJ, PARK, HONG and LIN2007b) investigated the effects of Pediococcus acidilactici and Saccharomyces boulardii-based probiotics on faecal oocyte shedding and serum antibody responses. They concluded that birds fed with 0.1% or 1.0% Pediococcus acidilactici and Saccharomyces boulardii-based probiotics decreased egg count in broiler birds infected with E. acervulina and E. tenella. They found that feeding a 0.1% Pediococcus acidilactici and Saccharomyces boulardii-based supplemented diet to E. acervulina infected birds resulted in higher serum eimeria-specific antibody titres compared to the control group.