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Comparison of the effects of three different types of probiotics on the Leucine Aminopeptidase (LAP) activities of the small intestine mucosa of broiler chicks.


In Greek Probiotic means "for life" (10). Probiotics are some Additives and Direct Fed-Microbial populations that can be added directly to food and balance intestinal micro-flora, microbial population can to some extent prevent intestinal infections, can have positive effect on animal performance and improve and increase growth of livestock.

Beneficial microflora promote gut development and health by influencing enterocyte turnover, competing with pathogenic bacteria for nutrients and binding sites, and producing bacteriostatic compounds that limit the growth of pathogenic bacteria (7). Probiotics have been defined as live microbial feed supplements, which beneficially affect bird by improving its intestinal microbial balance (8) probiotics are different from antibiotics, Microorganisms present in the probiotics are live and do not have certain chemical molecules. Probiotic does not have residual tissue and don't make any microbial resistance (10).

It has been used as a substitute of antibiotics that is being used inconsiderable amounts as growth promoters in broilers production and is, associated with incalculable risks for human health resulting from the use of particular feed additives.

Characteristics of an ideal probiotic for application in poultry nutrition

1. Should be capable of exerting a beneficial effect on the host animal, e.g. increase growth or increase resistance to disease.

2. Should be non-pathogenic & non toxic.

3. Inhibition of gram positive & gram negative germs (CE).

4. Should be present as viable cells, preferably in large numbers.

5. Should be capable of surviving & metabolizing in the gut environment, e.g. resistant to low pH & organic acids.

6. Should be stable & capable of remaining viable for long periods under storage & field conditions.

7. Not competitive for nutrient utilization [11].

The aim of this work was investigation on Effects of three Different Probiotics on the Leucine Aminopeptidase (LAP) enzyme activities in the small intestine mucosa of broilers.

Materials and methods

--Animal and Diets

180 male broilers of commercial hybrid (Ross 308) were designated into 4 groups. 3 replicates of 15 birds were assigned to each treatment. The birds were kept separately in cages next to each other and on the litter. All conditions for groups were the same except that control group was fed according to NRC Recommendations (1998) from a basal diet with no probiotic and the treatment groups were fed by basal diet containing three types of probiotic ([T.sub.1=Protexin] ,[T.sub.2=Bioplus 2B], [T.sub.3=Biosaf]). Diets were prepared according to NRC and during the first 21 days of rearing period and from 22 to 42 days by a starter and a finisher diet adlibitum (Table 1).

--Sample collection

In the Rearing period, all conditions such as temperature, humidity, light, ventilation and management were similar for all birds in treatments, according to standards. On day-21 and -42 of the rearing period, after 3-hours of fasting, 2 birds from each replicates (totally 24 chickens on each day of sampling) were removed and slaughtered. Samples of the small intestine were taken at 1, 10, 30, 50, 70 and 90 percent of the length for determining the LAP activities. They were rinsed in P.B.S pH#7 and after being wrapped in aluminum foil ,they were stored at -80 C.

--Enzyme assay

0.05 gram of the mucosa of the small intestine was weighed and along with 10 ml phosphate buffer saline (pH=7) using sonic Vibracell sonics device was homogenized. The LAP activity was measured according to Nigel et al., [14]. Total protein was also measured to convert its activity to IU in liter/gram protein [14].

--Statistical analysis

The results of the experiment was analyzed and by Multivariate Analysis of Variance by using the linear model of SPSS software [18]. Comparative analysis of the average of treatments was performed using ANOVAs multifunctional method and Tukey in the random of 5 percent.


According to Table 2, adding probiotic to the diet of the broilers at different ages and transferred to different parts of the small intestine caused variety of influences on the activity of LAP enzyme. In a way that the activity of LAP enzyme demonstrated a significant increase only at the age of 21 days after sampling as 1%,10% ,30% of the length of the small intestine in treatment groups comparing to control treatment was witnessed (P <0.05). probiotices, has significant effect on LAP activity In different ages and segments of small intestine.


In birds Microorganisms mostly colonize in the crop, caecum, and partly small intestine.

Those colonized in the small intestine are acquainted with their environment soon; they are mostly of the bacillus type. In order to settle for a long time, they have to stick to the surfaces of the villi, so it seems logical that they can produce some change on the small intestine villi [2]. Villi are more flat and leaf shape in herbivores, wheras in carnivores birds these villi are tall and finger shape [5,6] villi are covered with entrocytes which are responsible for absorption of food material [5]. Hampson (10) believes that measurements of the villus height and their shape can give us an indication of the enterocyte numbers [10]. Any change in villous height leads to a change in absorption rate. Leucine amino peptidase is one of the enzymes responsible for the hydrolysis of the proteins; they are present on the brush-border membrane of the mature enterocytes [17]. genetic expression of endopeptidases had been reported as early as 17 days in the mouse embryo and continued to increase by 19th day [13,16] It had been reported that using molds , as a probiotic To mice feed had increased brush--border enzyme activities like Sucrase , alkaline phosphatase and lecine amino peptidase [4, 12]. Adding probiotics to the diet According to table 2, in our work showed a significant elevation in leucine amino peptidase activity. In the proximal small intestine of broiler chicken By the end of the 3rd week Probiotics had affected the villous Dimension too so that both villous height and crypt Deep had increased and this is in agreement with our findings in the activities of leucine amino peptidase which is probably because an increase in the cell population therefore It can be concluded that probiotics can enhance the efficiency Of digestion and absorption mostly by the end of the first month of production in broilers.


[1.] Ahmad, I., 2006. Effect of Probiotics on Broilers Performance. International Journal of Poultry Science, 5(6): 593-597.

[2.] Angel, C.R., 1991. Long segmented filamentous organism observed in poults experimentally infected with stunting syn.

[3.] Benajiba, A. and S. Maroux, 1980. Purification and characterization. Eur. J. Biochem., 107: 381-388.

[4.] Buts, J.P., P. Bernasconi, M.P. Van Craynest, P. Maldague, M.R. De, 1986. Response of human and rat small intestinal mucosa to oral administration of Saccharomyces boulardii. Pediatr. Res., 20: 192-196.

[5.] Duke, G.E., 1986. Alimentary canal: anatomy. Regulation of feeding, and motility. In: Avian physiology. 4th ed., edited by P.D. Sturkie., Springer-Verlag, N.Y., pp: 269-88.

[6.] Duke, G.E., 1996. Avian Ddigestion. In: Dukes" physiology of domestic animals. 11th ed., edited by M.J. Swenson., and W.O. Reece. Cornell Univ. Press, Ithaca, N.Y., pp: 428-35.

[7.] Farthing, M.J.G., 2004. Bugs and the gut: An unstable marriage. Best Prac. Res. Clin. Gastroenterol., 18: 233-239.

[8.] Fuller, R., 1989. Probiotics in man and animals. J Appl Bacteriaol, 66: 365-378.

[9.] Gibson, G.R. and R. Fuller, 2000. Aspects of in vitro and in vivo research approaches directed toward identifying probiotics and prebiotics for human use. J. Nutr., 130: 391-395.

[10.] Hampson, D.J., 1986. Alterations in piglet small intestinal structure at weaning. Res. Vet. Science, 40: 32-40.

[11.] Hansen's.Chr., 2002. Guidelines for the Evaluation of Probiotics in Food: Joint FAO/WHO Working Group meeting, London Ontario, Canada, 30 April-1 May.

[12.] Jahn, H.U., R. Ullrich, T. Schneider, R.M. Liehr, H.L. Schieferdecker, H. Holst, M. Zeitz, 1996. Immunological and trophical effects of Saccharomyces boulardii on the small intestine in healthy human volunteers. Digestion, 57: 95-104.

[13.] Mouwen, J.M.V.M., 1971. White scours in piglets. Vet. Path., 8: 364-80.

[14.] Nagel, W., F. Willing, F.H. Schmidt, 1964. Fur die Aminosaurearylamidase (sog. Leucinaminopeptidase-) Aktivitat im menschlichen Serum. Klin. Wschr., 42: 447-9.

[15.] Palo, P.E., J.L. Sell, F.J. Piquer, L. Vilaseca and M.F. Soto Salanova, 1995. Effect of early nutrient restriction on broiler chickens. 2.Performance and digestive enzyme activities. Poult. Science, 74: 1470-1483.

[16.] Pavan, P.C., E.L. Ccrepaldi and J.B. Valim, 2000. Sorption of anionic surfactants on layered double hydroxides. J. Colloid and Interface Science, 229: 346-352.

[17.] Sklan, D., 2001. Development of the digestive tract of poultry. Worlds Poult. Science J., 57: 415-428.

[18.] SPSS Institute, 2002. SPSS state software: Changes and Enhancement through release 11.5.0, SPSS institute. LEAD Technologies. Inc, ALL RIGHTS RESERVED.

[19.] Watanaba, N., S. Kamel, A. Ohkubo, M. Yamanaka, S. Ohsaws, K. Maikino and K. Tokuda, 1986. Method for Assaying Total Protein. Clinical Chemistry, 32(8): 1551-1554.

Corresponding Author

Dr., Fazlollah moosavinasab, Ph.D of Veterinary physiology, Islamic Azad University, Science and Research Branch, Tehran, Iran Tel: +98 9161711172 E-mail:

(1) Fazlollah Moosavinasab, (1) Masoud Teshfam, (2) Jamshid Ghiasi ghaleh-kandi, (1) Babak Teshfam

(1) Islamic Azad University, Science and Research Branch--Tehran--Iran

(2) Islamic Azad University, Shabestar Branch, Shabestar--Iran

Fazlollah Moosavinasab, Masoud Teshfam, Jamshid Ghiasi ghaleh-kandi, BabakTeshfam; Comparison of the Effects of Three Different Types of Probiotics on the Leucine Aminopeptidase (LAP) Activities of the Small Intestine Mucosa of Broiler Chicks
Table 1: Ingredients and nutrient compositions of experimental diets

                       (1-21 Days)

                       0%           2%           4%


Corn                   54.5         54           45
SBM (%44)              34.14        34.19        35.81
Oil                    2.5          2.5          2.5
Methionine             0.6          0.6          0.8
Lysine                 0            0            0
Vitamin-premix         0.25         0.25         0.25
Mineral-premix         0.25         0.25         0.25
DCP                    1.6          1.6          1.62
Oyster                 1.44         1.4          1.33
Salt                   0.28         0.28         0.28
probiotic              0            2            4
Starch                 1.06         1.41         7.37
Fine Sand              3.38         1.46         0.07


ME (kcal/kg)           2850.21      2850.11      2850.14
Protein (Percent)      20.5         20.51        20.5
Calcium (Percent)      0.99         0.99         0.99
Phosphorus (Percent)   0.44         0.44         0.44
ME/Protein             139          138.96       139.03
Calcium/ Phosphorus    2.23         2.23         2.23

                       (21-42 Days)

                       0%           2%           4%


Corn                   62.64        39           39
SBM (%44)              27           27.7         27.7
Oil                    2.5          2.5          2.5
Methionine             0.6          0.6          0.6
Lysine                 0.2          0.2          0.2
Vitamin-premix         0.25         0.25         0.25
Mineral-premix         0.25         0.25         0.25
DCP                    1.13         1.13         1.13
Oyster                 1.48         1.44         1.44
Salt                   0.28         0.28         0.28
probiotic              0            2            2
Starch                 0            2.6          2.6
Fine Sand              3.67         2.05         2.05


ME (kcal/kg)           2920.54      2920.03      2920.03
Protein (Percent)      18.17        18.18        18.17
Calcium (Percent)      0.89         0.89         0.89
Phosphorus (Percent)   0.34         0.34         0.34
ME/Protein             160.69       160.64       160.64
Calcium/ Phosphorus    2.56         2.58         5.58

SBM = soybean meal; DCP = dicalcium phosphate. ME = Metabolisable
energy. Per 2.5 kg mineral supplement containing 99200 mg magnesium,
84700 mg zinc, 50000 mg iron, 10000 mg copper, 990 mg Iodine, 200 mg
selenium, 250000 ml gram Colin chloride. Per 2.5 kilogram vitamin
supplement containing 900000 IU of vitamin A, 200000 IU of vitamin
[D.sub.3], 19000 IU of vitamin E, 200 mg vitamin [K.sub.3], 18050 mg
vitamin [B.sub.1], 49000 mg vitamin [B.sub.2], 9800 mg vitamin
[B.sub.3], 29650 mg vitamin [B.sub.5], 2940 mg vitamin [B.sub.6],
1000 mg vitamin [B.sub.9], 15 mg vitamin [B.sub.12], 100 mg biotin,
190000 mg cholin chloride, 1000 mg antioxidant.

Table 2: Comparison of average LAP activity between treatment and
control groups in different periods and segments of small intestine
in broiler chicks (IU/g protein)

age    Different Type of % length of small intestine

                         1%               10%

21     control (a)       1676.8494abcd    1605.1109abd
       protoxin (b)      1830.2470abd     1671.2261abd
       Bioplus 2B (c)    1559.0645acd     1054.5540c
       biosaf (d)        1626.5957abcd    1707.9546abd

42     control (a)       1871.2804abcd    1421.1538abcd
       protoxin (b)      1699.2841abcd    1643.3408abcd
       Bioplus 2B (c)    1652.9103abcd    1848.0100abcd
       biosaf (d)        1726.8167abcd    1442.3398abcd

age    Different Type of % length of small intestine

                         30%              50%

21     control (a)       1635.1348abc     1313.0505abcd
       protoxin (b)      1501.2807abd     1504.3251abcd
       Bioplus 2B (c)    1543.5012abcd    1505.9089abcd
       biosaf (d)        1171.2592cd      1700.4511abcd

42     control (a)       1645.2440abcd    2032.2272abcd
       protoxin (b)      1410.2897abcd    1853.2390abcd
       Bioplus 2B (c)    1859.5133abcd    1548.0352abcd
       biosaf (d)        1257.6544abcd    1641.6667abcd

age    Different Type of % length of small intestine

                         70%              90%

21     control (a)       1493.8130abcd    1581.3052abcd
       protoxin (b)      1839.7866abcd    1405.7866abcd
       Bioplus 2B (c)    1523.1595abcd    1687.6261abcd
       biosaf (d)        1304.7956abcd    1601.5063abcd

42     control (a)       1751.0692abcd    1999.5342abcd
       protoxin (b)      1536.6959abcd    1887.3166abcd
       Bioplus 2B (c)    1149.6520abcd    1909.3707abcd
       biosaf (d)        1606.5590abcd    1709.0851abcd

* Unsimilar sign letters show significant differences (P <0.05)
between given means.
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Article Details
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Title Annotation:Original Article
Author:Moosavinasab, Fazlollah; Teshfam, Masoud; Ghaleh-kandi, Jamshid Ghiasi; Teshfam, Babak
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7IRAN
Date:Jan 1, 2011
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