Probiotics & Antimicro. Prot. (2014) 6:136–140 DOI 10.1007/s12602-014-9170-6
Bone Mineralization of Broiler Chicks Challenged with Salmonella enteritidis Fed Diet Containing Probiotic (Bacillus subtilis) A. A. Sadeghi
Published online: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study was carried out to determine the effect of probiotic, Bacillus subtilis, on ash and calcium contents of tibia bone in unchallenged and challenged broiler chicks with Salmonella enteritidis. In a completely randomized design, 160 chicks were divided into four groups. Each group had four replicates with 10 birds each. Treatments were control group, probiotic-treated group, challenged group and challenged probiotic-treated group. Ash and calcium contents of tibia at 21 and 42 days of age were determined. At 21 days of age, the highest contents of ash and calcium were related to probiotic-treated group and the lowest means to challenged chicks (P \ 0.05). At this period, inclusion of probiotic to diet of challenged chick increased (P \ 0.05) ash and calcium contents of tibia. With increases in age, the negative effects of challenging and beneficial effects of probiotic on bone mineralization diminished; since at 42 days of age, challenging or probiotic treatment had no effect on ash and calcium contents of tibia.
bone, and ash or inorganic substances such as calcium and phosphorus, which provide strength and hardness [1]. Development and mineralization of normal bone in broiler chicks are influenced by many factors, including feed additives such as probiotics and infections [2]. Several studies in animals and humans have shown the negative effect of infection [3] and positive effect of probiotics [3–5] on mineral absorption and metabolism and bone composition and architecture. Despite the fact that several studies have shown bone development enhancement resulting from oral administration of probiotics or infection in mammalians, there is a dearth of information regarding the effect of probiotic or infection, alone or in combination, on the bone mineralization of broiler chicks. Therefore, the present study was carried out to determine the effect of probiotic, Bacillus subtilis, on ash and calcium contents of tibia bone in unchallenged chicks or those challenged with Salmonella enteritidis.
Keywords Probiotic Salmonella Bone mineralization Tibia Broiler chicks
Materials and Methods Preparation of Probiotic Sample and Salmonella Inoculum
Introduction Several metabolic problems are more pronounced in fastgrowing male broilers, including skeletal disorders. The skeleton is an important mineral source for metabolic needs; in addition it provides structural support for the bird. Bone tissue is composed of organic substances, which give elasticity to A. A. Sadeghi (&) Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran e-mail: [email protected]
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The probiotic was obtained from Iranian Baltec Company (presentation of Biochem, Lohne, Germany) and included in the diets at the amount of 0.20 g/kg (0.02 % of dry matter). Salmonella enteritidis (PTCC 1709) was obtained as freeze-dried from the Persian Type Culture Collection (IROST, Tehran, Iran) isolated from the liver of chickens. Salmonella Culturing and Challenging Freeze-dried inoculum was added and grown in tryptic soy broth (Acumedia Manufacturers Inc., Baltimore, MD, USA)
Probiotics & Antimicro. Prot. (2014) 6:136–140
137
Table 1 Ingredients and chemical composition of experimental rations (On an as-fed basis) Ingredients (%)
Starter
Grower
Finisher
Yellow corn
59.12
62.31
63.84
Soybean meal (44 % CP)
32.62
30.09
29.13
Fish meal
3.00
2.00
–
Soybean oil
1.71
2.42
3.87
DCP
1.53
1.43
1.48
CaCO3
1.10
1.05
1.03
Salt Mineral premix
0.36 0.10
0.24 0.10
0.18 0.10
Vitamin premix
0.25
0.25
0.25
Methionine
0.17
0.03
0.05
Lysine
–
0.05
0.05
Probiotic
0.02
0.02
0.02
ME (kcal/kg)
2,920
3,000
3,100
Protein (%)
21.0
19.5
18.00
Calcium (%)
1.00
0.90
0.80
Phosphorus (%)
0.5
0.45
0.40
Chemical composition
at 37 °C for 8 h, then subcultured on fresh tryptic soy broth for 3 incubation periods. Colony-forming unit (cfu) method was used to calculate the number of viable bacteria in Salmonella enteritidis suspension by 0.9 % peptone water (pH 7.2). For this purpose, 0.1 ml of the bacteria suspension was inoculated in Petri dishes containing Shigella–Salmonella agar (SS agar) and incubated for 24 h at 37 °C, then cfu counted. At day 7, chicks in challenged groups received by oral gavage 1.0 9 105 cfu per chick (0.2 ml) of Salmonella enteritidis suspension. Unchallenged birds received the same amount of sterile-buffered peptone water by oral gavage. Birds and Experimental Design One hundred and sixty 1-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery. At a completely randomized design, the birds were randomly divided into four groups and housed in isolated pens of identical size (1.3 9 1.2 m) in a litter of wood shavings. Each group had four replicates with 10 birds each. The treatments were (1) control group (basal diet without probiotic supplementation and challenging), (2) probiotictreated group (basal diet supplemented with 0.20 g probiotic per kg of diet, without pathogen challenge), (3) challenged group (basal diet, with pathogen challenge at 7 days of age) and (4) challenged probiotic-treated group (basal diet supplemented with probiotic and pathogen challenge at 7 days of age). Basal diet was formulated based on corn– soybean meal without enzyme and antibiotic
supplementations (Table 1). Experimental diets were analyzed for ash, crude protein, calcium, and phosphorus according to the methods of AOAC [6]. Birds had free access to water. Environmental temperature in the first week of life was 33 °C and decreased to 20 °C until the end of the experiment. During the first week, 22 h of light was provided with a reduction to 20 h afterward. At 30 days of age, litter moisture contents were determined in each pen by collecting three samples (300 g) in areas of the pen away from feeders and drinkers. The three samples were mixed together and one homogeneous subsample was weighed (50 g), oven-dried at 105 °C for 24 h, and reweighed to determine moisture content. Ash and Calcium Determination At 21 and 42 days of age, eight chickens per treatment (two chickens per pen) were randomly selected and killed by cervical dislocation. To determine the percentage of tibia ash, the right leg was removed as processed, following the procedure described by Hall et al. [7]. Briefly, the tibias of killed broilers were boiled to remove the tissues and cartilage caps. Ground-dried tibia samples were extracted to remove the fat using Soxhlet apparatus. Then, the extracted samples were burned on an electric heater and placed in a muffle furnace at 550 °C for 5 h and ash weight of each sample was expressed as the percent of dry-extracted bone weight. Calcium concentration was measured by atomic absorption spectrometry. Statistical Analysis Statistical analyses were conducted with the general linear model procedure of SAS for Windows, version 9.1 (SAS Institute Inc., Cary, NC) to determine whether variables differed between groups. The Kolmogorov–Smirnov test was used to test the normal distribution of the data before statistical analysis was performed. The data were compared between groups by Tukey’s test. Probability values of less than 0.05 (P \ 0.05) were considered significant.
Results Effects on Litter Moisture and Survivability Effects of challenging and probiotic treatment on moisture content of litter at 30 days of age and survivability of chicks are given in Table 2. The lowest litter moisture was related to probiotic-treated group and highest to challenged chicks. Litter moisture content decreased (P \ 0.05) in challenged probiotic-treated chicks as compared with that of challenged group.
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Probiotics & Antimicro. Prot. (2014) 6:136–140
Table 2 Moisture content of litter and survivability of chicks Treatments
Litter moisture content (%)
Survivability (%)
Control
48.6a
85a
c
88a
a
70b
b
83a
Probiotic treated Challenged Challenged and probiotic treated
22.1 55.2
34.4
SEM
2.82
4.03
SEM standard error of the means a,b,c
Means in the same column with different superscripts are differ (P \ 0.05)
Fig. 2 Calcium content of tibia at 21 days of age
Fig. 1 Ash content of tibia at 21 days of age
The highest mean of survivability was recorded in the probiotic-treated group and lowest in the challenged chicks. Dietary inclusion of probiotic to diet of challenged chicks increased (P \ 0.05) survivability of chicks.
Fig. 3 Ash content of tibia at 42 days of age
Discussion Effects on Ash and Calcium Contents of Tibia Effects of challenging and probiotic treatment on ash and calcium contents of tibia at 21 and 42 days of age are in Figs. 1, 2, 3, and 4. At 21 days of age, the highest ash and calcium contents of tibia were related to probiotic-treated group and lowest means to challenged chicks. The percentages of ash and calcium contents of tibia improved (P \ 0.05) by dietary probiotic supplementation. At 42 days of age, there was no significant effect of challenging or probiotic treatment on ash and calcium contents of tibia.
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The present study was designed to evaluate the effects of dietary inclusion of probiotic (Bacillus subtilis) on tibia ash and calcium contents of Salmonella-challenged broiler chicks. It is hypothesized that Salmonella challenging would decrease the bone mineralization and inclusion of probiotic to diet of unchallenged and challenged chicks would enhance it. Moisture content of litter in challenged chicks was higher than unchallenged ones, which indicates a development of infection through challenging program.
Probiotics & Antimicro. Prot. (2014) 6:136–140
Fig. 4 Calcium content of tibia at 42 days of age
Mortality in challenged groups increased 3 days after Salmonella infection. Mortality was observed from the third day of post-challenge which was confirmed due to the challenge infection with Salmonella enteritidis. The mortality was observed up to 10 days. Chicks before dead were weak, lethargic with distinct green diarrhea. Dietary inclusion of probiotic decreased litter moisture content and mortality of challenged chicks, significantly, which indicates its reducing effect on Salmonella colonization in intestine. At 21 days of age, ash and calcium contents of tibia in probiotic-treated group were numerically higher than that in control group. Our results are in agreement with Nahashon et al. [4] and Panda et al. [5]. Nahashon et al. [4] reported positive correlations between dietary inclusion of Lactobacillus and calcium retention. Panda et al. [6] suggested that Lactobacillus sporogenes has a positive effect on tibia ash and also on resistance to fracture due to more retention of calcium in bones. In fact, supplementation of a diet with probiotic could lead to a more magnitude of serum calcium associating with establishing a more appropriate absorption environment in digestive tract. Also, Plavnik and Scott [8] reported that chickens receiving probiotic-based brewer’s yeast had much lower incidence of tibial dyschondroplasia and bone strength was increased by the dietary probiotic supplementation. They speculated that higher than normal retained calcium might have been deposited in the skeleton. Results of this study showed that Salmonella challenging resulted in decrease in ash and calcium contents of tibia bone. Bone is a living tissue that is under constant reconstruction through an intricate system that facilitates bone
139
resorption by osteoclasts and bone formation by osteoblasts. Bone resorption is regulated through increased expression of receptor activator of NF-kB ligand (RANKL) [9] and its receptor. It has been shown that microorganisms such as Staphylococcus aureus and Salmonella induce the expression of receptor activator of RANKL in osteoblastlike cells and synovial fibroblasts mediating osteoclast activation [10–12]. Bone resorption is increased through increased expression of RANKL [9]. Other cytokines such as various interleukins and tumor necrosis factor alpha (TNF-alpha) can also influence this balance [9]. It has been shown that Salmonella stimulate different subsets of immune cells to produce cytokines, especially interleukin-1 b [13]. Interleukin-1 is a multi-functional, pro-inflammatory cytokine that has been associated with bone loss in human and as a mediator of bone loss in rheumatoid arthritis [3]. The addition of probiotic to diet of challenged chicks increased significantly ash and calcium contents of tibia. There is no study found in the literature, in which the effects of probiotic on ash and calcium contents of tibia in challenged chicks were evaluated. The two most likely mechanisms by which probiotic reduce the negative effects of Salmonella on many parameters, especially bone mineralization, involve competitive exclusion [14]. The Bacillus subtilis could reduce Salmonella counts in intestines through a number of these inhibitory mechanisms. Recently, an interesting study [15] in human revealed that oral administration of Bacillus subtilis could reduce proinflammatory cytokines (interleukin-1, TNF-alpha, interleukin-6, and Interferon-c) and increase anti-inflammatory cytokine (interleukin-10 and TGF-beta) in plasma of patients with inflammatory bowel disease. These events may reduce the negative effects of Salmonella on bone mineralization of chicks in this study. Probiotics for poultry have been extensively reviewed by Jin et al. [16], Simmering and Blaut [17], and Patterson and Burkholder [18]. According to these review articles, it is concluded that there has not been a well-established link between microbial probiotics and mineral absorption or bone development. At 42 days of age, challenging or probiotic treatment had no effect on ash and calcium contents of tibia. It seems that with increases in age, the negative effects of Salmonella challenging and beneficial effects of probiotic supplementation reduce. The contradictions among results of various studies concerning effect of feed additives or other treatments on bone mineralization may be related to age of animals used in the experiments. In the future studies, considering age of experimental animals as an effective factor on bone mineralization for comparison and evaluating the feed additive or infection is recommended.
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Probiotics & Antimicro. Prot. (2014) 6:136–140
The present study demonstrated that ash and calcium contents of the tibia of broilers at early age are affected when fed probiotic-supplemented diets or infected with Salmonella. Inclusion of probiotic (Bacillus subtilis) to diet of challenged chicks compensated the negative effects of Salmonella challenging on bone mineralization. With increases in age, the negative effects of challenging and beneficial effects of probiotic on bone mineralization diminished. There is a need for further studies to determine the effects and mode of action of probiotics on bone development and mineralization with considering age effects.
6. 7.
8.
9.
Acknowledgments The author is grateful to the Islamic Azad University, Science and Research Branch for research funding support and to Agricultural, Medical and Industrial Research School, NSTR (AEOI) for supplying the basal diet and allowing use of poultry unit and to Baltec Company (presentation of Biochem Company, Germany) for providing probiotic Gallipro sample. The author also thanks all staffs (Mr. Zargaran and Mrs. Shakorzadeh) in the poultry unit for the assistance in the care and feeding of the chicks used in this research.
10.
The author declares that he has no conflict of
13.
Conflict of interest interest.
References 1. Mutus R, Kocabagli N, Alp M, Acar N, Eren M, Gezen SS (2006) The effect of dietary probiotic supplementation on tibial bone characteristics and strength in broilers. Poult Sci 85:1621–1625 2. Warren KR, Neuman WF (1971) Some factors affecting mineralization of bone in tissue culture. Am J Physiol 220:270–274 3. van Heeckeren AM, Rikihisa Y, Park J, Fertel R (1993) Tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, and prostaglandin E2 production in murine peritoneal macrophages infected with Ehrlichia risticii. Infect Immun 61:4333–4337 4. Nahashon SN, Nakaue HS, Mirosh LW (1994) Production variable and nutrient retention in Single Comb White Leghorn laying pullets fed diets supplemented with direct fed microbials. Poult Sci 73:1699–1711 5. Panda AK, Rama Rao SV, Raju MVLN, Sharma SR (2006) Dietary supplementation of Lactobacillus sporgenes on
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performance and serum biochemico-lipid profile of broiler chickens. J Poult Sci 43:235–240 AOAC (1995) Official methods of analysis, 16th ed. Association of Official Analytical Chemists. Arlington, Virginia, USA Hall LE, Shirley RB, Bakalli RI, Aggrey SE, Pesti GM, Edwards HM (2003) Power of two methods for the estimation of bone ash of broilers. Poult Sci 82:414–418 Plavnik I, Scott ML (1980) Effects of additional vitamins, minerals or brewer’s yeast upon leg weaknesses in broiler chickens. Poult Sci 59:459–464 Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senald G, Guo J, Delaney J, Boyle WJ (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176 Marriott I (2004) Osteoblast responses to bacterial pathogens: a previously unappreciated role for bone-forming cells in host defense and disease progression. Immunol Res 30:291–308 Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, Penninger JM (2000) Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clinic Invest 106:R59–R67 Henderson B, Nair SP, Ward JM, Wilson M (2003) Molecular pathogenicity of the oral opportunistic pathogen Actinobacillus actinomycetemcomitans. Annu Rev Microbiol 57:29–55 Okamura M, Lillehoj HS, Raybourne RB, Babu US, Heckert RA (2004) Cell-mediated immune responses to a killed Salmonella enteritidis vaccine: lymphocyte proliferation, T-cell changes and interleukin-6 (IL-6), IL-1, IL-2, and IFN-y production. Comp Immunol Microbiol Infect Dis 27:255–272 Gaggia F, Mattarelli P, Biavati B (2010) Probiotics and prebiotics in animal feeding for safe food production. Inter J Food Microbiol 141:S15–S28 Selvam R, Maheswari P, Kavitha P, Ravichandran M, Sas B, Ramchand CN (2009) Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease. Ind J Biochem Biophys 46:79–85 Jin LZ, Ho YW, Abdullah N, Jalaludin S (1997) Probiotics in poultry: modes of action. Worlds Poult Sci J 53:351–368 Simmering R, Blaut M (2001) Pro- and prebiotics: the tasty guardian angels? Appl Microbiol Biotechnol 55:19–28 Patterson JA, Burkholder KM (2003) Application of prebiotics and probiotics in poultry production. Poult Sci 82:627–631
Bone Mineralization of Broiler Chicks Challenged with Salmonella enteritidis Fed Diet Containing Probiotic (Bacillus subtilis) A. A. Sadeghi
Published online: 10 October 2014 Ó Springer Science+Business Media New York 2014
Abstract The present study was carried out to determine the effect of probiotic, Bacillus subtilis, on ash and calcium contents of tibia bone in unchallenged and challenged broiler chicks with Salmonella enteritidis. In a completely randomized design, 160 chicks were divided into four groups. Each group had four replicates with 10 birds each. Treatments were control group, probiotic-treated group, challenged group and challenged probiotic-treated group. Ash and calcium contents of tibia at 21 and 42 days of age were determined. At 21 days of age, the highest contents of ash and calcium were related to probiotic-treated group and the lowest means to challenged chicks (P \ 0.05). At this period, inclusion of probiotic to diet of challenged chick increased (P \ 0.05) ash and calcium contents of tibia. With increases in age, the negative effects of challenging and beneficial effects of probiotic on bone mineralization diminished; since at 42 days of age, challenging or probiotic treatment had no effect on ash and calcium contents of tibia.
bone, and ash or inorganic substances such as calcium and phosphorus, which provide strength and hardness [1]. Development and mineralization of normal bone in broiler chicks are influenced by many factors, including feed additives such as probiotics and infections [2]. Several studies in animals and humans have shown the negative effect of infection [3] and positive effect of probiotics [3–5] on mineral absorption and metabolism and bone composition and architecture. Despite the fact that several studies have shown bone development enhancement resulting from oral administration of probiotics or infection in mammalians, there is a dearth of information regarding the effect of probiotic or infection, alone or in combination, on the bone mineralization of broiler chicks. Therefore, the present study was carried out to determine the effect of probiotic, Bacillus subtilis, on ash and calcium contents of tibia bone in unchallenged chicks or those challenged with Salmonella enteritidis.
Keywords Probiotic Salmonella Bone mineralization Tibia Broiler chicks
Materials and Methods Preparation of Probiotic Sample and Salmonella Inoculum
Introduction Several metabolic problems are more pronounced in fastgrowing male broilers, including skeletal disorders. The skeleton is an important mineral source for metabolic needs; in addition it provides structural support for the bird. Bone tissue is composed of organic substances, which give elasticity to A. A. Sadeghi (&) Department of Animal Science, Science and Research Branch, Islamic Azad University, Tehran, Iran e-mail: [email protected]
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The probiotic was obtained from Iranian Baltec Company (presentation of Biochem, Lohne, Germany) and included in the diets at the amount of 0.20 g/kg (0.02 % of dry matter). Salmonella enteritidis (PTCC 1709) was obtained as freeze-dried from the Persian Type Culture Collection (IROST, Tehran, Iran) isolated from the liver of chickens. Salmonella Culturing and Challenging Freeze-dried inoculum was added and grown in tryptic soy broth (Acumedia Manufacturers Inc., Baltimore, MD, USA)
Probiotics & Antimicro. Prot. (2014) 6:136–140
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Table 1 Ingredients and chemical composition of experimental rations (On an as-fed basis) Ingredients (%)
Starter
Grower
Finisher
Yellow corn
59.12
62.31
63.84
Soybean meal (44 % CP)
32.62
30.09
29.13
Fish meal
3.00
2.00
–
Soybean oil
1.71
2.42
3.87
DCP
1.53
1.43
1.48
CaCO3
1.10
1.05
1.03
Salt Mineral premix
0.36 0.10
0.24 0.10
0.18 0.10
Vitamin premix
0.25
0.25
0.25
Methionine
0.17
0.03
0.05
Lysine
–
0.05
0.05
Probiotic
0.02
0.02
0.02
ME (kcal/kg)
2,920
3,000
3,100
Protein (%)
21.0
19.5
18.00
Calcium (%)
1.00
0.90
0.80
Phosphorus (%)
0.5
0.45
0.40
Chemical composition
at 37 °C for 8 h, then subcultured on fresh tryptic soy broth for 3 incubation periods. Colony-forming unit (cfu) method was used to calculate the number of viable bacteria in Salmonella enteritidis suspension by 0.9 % peptone water (pH 7.2). For this purpose, 0.1 ml of the bacteria suspension was inoculated in Petri dishes containing Shigella–Salmonella agar (SS agar) and incubated for 24 h at 37 °C, then cfu counted. At day 7, chicks in challenged groups received by oral gavage 1.0 9 105 cfu per chick (0.2 ml) of Salmonella enteritidis suspension. Unchallenged birds received the same amount of sterile-buffered peptone water by oral gavage. Birds and Experimental Design One hundred and sixty 1-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery. At a completely randomized design, the birds were randomly divided into four groups and housed in isolated pens of identical size (1.3 9 1.2 m) in a litter of wood shavings. Each group had four replicates with 10 birds each. The treatments were (1) control group (basal diet without probiotic supplementation and challenging), (2) probiotictreated group (basal diet supplemented with 0.20 g probiotic per kg of diet, without pathogen challenge), (3) challenged group (basal diet, with pathogen challenge at 7 days of age) and (4) challenged probiotic-treated group (basal diet supplemented with probiotic and pathogen challenge at 7 days of age). Basal diet was formulated based on corn– soybean meal without enzyme and antibiotic
supplementations (Table 1). Experimental diets were analyzed for ash, crude protein, calcium, and phosphorus according to the methods of AOAC [6]. Birds had free access to water. Environmental temperature in the first week of life was 33 °C and decreased to 20 °C until the end of the experiment. During the first week, 22 h of light was provided with a reduction to 20 h afterward. At 30 days of age, litter moisture contents were determined in each pen by collecting three samples (300 g) in areas of the pen away from feeders and drinkers. The three samples were mixed together and one homogeneous subsample was weighed (50 g), oven-dried at 105 °C for 24 h, and reweighed to determine moisture content. Ash and Calcium Determination At 21 and 42 days of age, eight chickens per treatment (two chickens per pen) were randomly selected and killed by cervical dislocation. To determine the percentage of tibia ash, the right leg was removed as processed, following the procedure described by Hall et al. [7]. Briefly, the tibias of killed broilers were boiled to remove the tissues and cartilage caps. Ground-dried tibia samples were extracted to remove the fat using Soxhlet apparatus. Then, the extracted samples were burned on an electric heater and placed in a muffle furnace at 550 °C for 5 h and ash weight of each sample was expressed as the percent of dry-extracted bone weight. Calcium concentration was measured by atomic absorption spectrometry. Statistical Analysis Statistical analyses were conducted with the general linear model procedure of SAS for Windows, version 9.1 (SAS Institute Inc., Cary, NC) to determine whether variables differed between groups. The Kolmogorov–Smirnov test was used to test the normal distribution of the data before statistical analysis was performed. The data were compared between groups by Tukey’s test. Probability values of less than 0.05 (P \ 0.05) were considered significant.
Results Effects on Litter Moisture and Survivability Effects of challenging and probiotic treatment on moisture content of litter at 30 days of age and survivability of chicks are given in Table 2. The lowest litter moisture was related to probiotic-treated group and highest to challenged chicks. Litter moisture content decreased (P \ 0.05) in challenged probiotic-treated chicks as compared with that of challenged group.
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Table 2 Moisture content of litter and survivability of chicks Treatments
Litter moisture content (%)
Survivability (%)
Control
48.6a
85a
c
88a
a
70b
b
83a
Probiotic treated Challenged Challenged and probiotic treated
22.1 55.2
34.4
SEM
2.82
4.03
SEM standard error of the means a,b,c
Means in the same column with different superscripts are differ (P \ 0.05)
Fig. 2 Calcium content of tibia at 21 days of age
Fig. 1 Ash content of tibia at 21 days of age
The highest mean of survivability was recorded in the probiotic-treated group and lowest in the challenged chicks. Dietary inclusion of probiotic to diet of challenged chicks increased (P \ 0.05) survivability of chicks.
Fig. 3 Ash content of tibia at 42 days of age
Discussion Effects on Ash and Calcium Contents of Tibia Effects of challenging and probiotic treatment on ash and calcium contents of tibia at 21 and 42 days of age are in Figs. 1, 2, 3, and 4. At 21 days of age, the highest ash and calcium contents of tibia were related to probiotic-treated group and lowest means to challenged chicks. The percentages of ash and calcium contents of tibia improved (P \ 0.05) by dietary probiotic supplementation. At 42 days of age, there was no significant effect of challenging or probiotic treatment on ash and calcium contents of tibia.
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The present study was designed to evaluate the effects of dietary inclusion of probiotic (Bacillus subtilis) on tibia ash and calcium contents of Salmonella-challenged broiler chicks. It is hypothesized that Salmonella challenging would decrease the bone mineralization and inclusion of probiotic to diet of unchallenged and challenged chicks would enhance it. Moisture content of litter in challenged chicks was higher than unchallenged ones, which indicates a development of infection through challenging program.
Probiotics & Antimicro. Prot. (2014) 6:136–140
Fig. 4 Calcium content of tibia at 42 days of age
Mortality in challenged groups increased 3 days after Salmonella infection. Mortality was observed from the third day of post-challenge which was confirmed due to the challenge infection with Salmonella enteritidis. The mortality was observed up to 10 days. Chicks before dead were weak, lethargic with distinct green diarrhea. Dietary inclusion of probiotic decreased litter moisture content and mortality of challenged chicks, significantly, which indicates its reducing effect on Salmonella colonization in intestine. At 21 days of age, ash and calcium contents of tibia in probiotic-treated group were numerically higher than that in control group. Our results are in agreement with Nahashon et al. [4] and Panda et al. [5]. Nahashon et al. [4] reported positive correlations between dietary inclusion of Lactobacillus and calcium retention. Panda et al. [6] suggested that Lactobacillus sporogenes has a positive effect on tibia ash and also on resistance to fracture due to more retention of calcium in bones. In fact, supplementation of a diet with probiotic could lead to a more magnitude of serum calcium associating with establishing a more appropriate absorption environment in digestive tract. Also, Plavnik and Scott [8] reported that chickens receiving probiotic-based brewer’s yeast had much lower incidence of tibial dyschondroplasia and bone strength was increased by the dietary probiotic supplementation. They speculated that higher than normal retained calcium might have been deposited in the skeleton. Results of this study showed that Salmonella challenging resulted in decrease in ash and calcium contents of tibia bone. Bone is a living tissue that is under constant reconstruction through an intricate system that facilitates bone
139
resorption by osteoclasts and bone formation by osteoblasts. Bone resorption is regulated through increased expression of receptor activator of NF-kB ligand (RANKL) [9] and its receptor. It has been shown that microorganisms such as Staphylococcus aureus and Salmonella induce the expression of receptor activator of RANKL in osteoblastlike cells and synovial fibroblasts mediating osteoclast activation [10–12]. Bone resorption is increased through increased expression of RANKL [9]. Other cytokines such as various interleukins and tumor necrosis factor alpha (TNF-alpha) can also influence this balance [9]. It has been shown that Salmonella stimulate different subsets of immune cells to produce cytokines, especially interleukin-1 b [13]. Interleukin-1 is a multi-functional, pro-inflammatory cytokine that has been associated with bone loss in human and as a mediator of bone loss in rheumatoid arthritis [3]. The addition of probiotic to diet of challenged chicks increased significantly ash and calcium contents of tibia. There is no study found in the literature, in which the effects of probiotic on ash and calcium contents of tibia in challenged chicks were evaluated. The two most likely mechanisms by which probiotic reduce the negative effects of Salmonella on many parameters, especially bone mineralization, involve competitive exclusion [14]. The Bacillus subtilis could reduce Salmonella counts in intestines through a number of these inhibitory mechanisms. Recently, an interesting study [15] in human revealed that oral administration of Bacillus subtilis could reduce proinflammatory cytokines (interleukin-1, TNF-alpha, interleukin-6, and Interferon-c) and increase anti-inflammatory cytokine (interleukin-10 and TGF-beta) in plasma of patients with inflammatory bowel disease. These events may reduce the negative effects of Salmonella on bone mineralization of chicks in this study. Probiotics for poultry have been extensively reviewed by Jin et al. [16], Simmering and Blaut [17], and Patterson and Burkholder [18]. According to these review articles, it is concluded that there has not been a well-established link between microbial probiotics and mineral absorption or bone development. At 42 days of age, challenging or probiotic treatment had no effect on ash and calcium contents of tibia. It seems that with increases in age, the negative effects of Salmonella challenging and beneficial effects of probiotic supplementation reduce. The contradictions among results of various studies concerning effect of feed additives or other treatments on bone mineralization may be related to age of animals used in the experiments. In the future studies, considering age of experimental animals as an effective factor on bone mineralization for comparison and evaluating the feed additive or infection is recommended.
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The present study demonstrated that ash and calcium contents of the tibia of broilers at early age are affected when fed probiotic-supplemented diets or infected with Salmonella. Inclusion of probiotic (Bacillus subtilis) to diet of challenged chicks compensated the negative effects of Salmonella challenging on bone mineralization. With increases in age, the negative effects of challenging and beneficial effects of probiotic on bone mineralization diminished. There is a need for further studies to determine the effects and mode of action of probiotics on bone development and mineralization with considering age effects.
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Acknowledgments The author is grateful to the Islamic Azad University, Science and Research Branch for research funding support and to Agricultural, Medical and Industrial Research School, NSTR (AEOI) for supplying the basal diet and allowing use of poultry unit and to Baltec Company (presentation of Biochem Company, Germany) for providing probiotic Gallipro sample. The author also thanks all staffs (Mr. Zargaran and Mrs. Shakorzadeh) in the poultry unit for the assistance in the care and feeding of the chicks used in this research.
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The author declares that he has no conflict of
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Conflict of interest interest.
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