Livestock Research for Rural Development 19 (11) 2007 Guide for preparation of papers LRRD News

Citation of this paper

Effect of a combination of organic acid salts as antibiotic replacer on the performance and gut health of broiler chickens

S K Paul, G Samanta, G Halder and P Biswas

Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences
37, Kshudiram Bose Sarani, Kolkata- 700 037, India
samik72@rediffmail.com

Abstract

An experiment was conducted to determine the influence of a combination of organic acid salts as substitute for antibiotics on the performance and gut health of broiler chickens. One hundred and twenty day-old commercial Vencobb-100 broiler chicks were purchased and randomly distributed into three groups, with four replicates of 10 birds in each group. Control (C) birds were given a standard basal diet with virginiamycin @ 0.5 g/kg as antibiotic; Treatment 1 (T1) was a diet with 1.5g/kg each of ammonium formate and calcium propionate and Treatment 2 (T2) a diet with 1g/kg each of calcium propionate, ammonium formate and calcium lactate.

 

No significant difference between antibiotic and the acidifier supplemented groups was found in live weight or live weight gain (LWG). However, cumulative feed intake was higher (P<0.05) in the antibiotic supplemented group compared with the acidifier supplemented groups. Supplementation of organic acid salts significantly improved (P<0.05) feed conversion ratio (FCR) compared with the antibiotic treatment. Addition of antibiotic and acidifier did not have any significant effect on coliform and clostridium count in the feed, whereas organic acid salts reduced the fungal count. Escherichia coli and clostridium counts in the gut varied numerically among treatments, but the differences were not statistically significant. Propionate, formate and lactate supplementation improved (P<0.05) duodenal villus height, which was also improved by acidifier treatment. No change (P>0.05) in pH of different segments of the G.I. tract was observed between the treatments.

Key words: Broiler chickens, gut health, organic acid salts, performance


Introduction

With the declining use of antibiotic growth promoters due to the development of antibiotic-resistant strains of pathogenic microorganisms, especially gram-negative, there is an ongoing need to identify effective replacements. Organic acids in their undissociated forms are able to pass through the cell membranes of bacteria, where the acids dissociate in an alkaline medium to produce H+ ions which lower the pH of the cell, causing the organism to use its energy in trying to restore the normal balance. The RCOOanions produced from the acid can disrupt DNA and protein synthesis, putting the organism under stress, so that it is unable to replicate rapidly (Nursey 1997). However, lactic acid bacteria are able to grow at relatively low pH, which means that they are more resistant to organic acids than other bacterial species. An explanation may be that gram positive bacteria have a high intracellular K+ concentration, which provides a counteraction for the acid anions (Russell and Diez-Gonzalez 1998). Generally two types of acidifiers are used, feed acidifiers and gut acidifiers. Feed acidifiers are pure organic acids which can be added to the feed by direct spraying. However, pure organic acids corrode the G.I. tract of poultry, and also are difficult to handle.  Gut acidifiers are organic acid salts, such as ammonium di-propionate, potassium di-formate, sodium formate, calcium propionate, calcium lactate, ammonium formate etc., and have little or no corrosive effect on the G.I. tract of poultry. Thus the objective of the present study was to determine the effect of a combination of organic acid salts as an antibiotic substitute on the performance and gut health of broiler chickens.
 

 

Materials and methods

 

Birds and experimental design

 

A total of 120 day-old Vencobb-100 broiler chicks were used in the experiment. The chicks were individually weighed and allocated to 12 cages of 10 birds each, so that average initial body weight of birds of each cage did not vary significantly (P>0.05). Birds were randomly allotted to three treatment groups, each with four replicates of ten birds.  Control (C) birds were given a standard basal diet with virginiamycin @ 0.5 g/kg as antibiotic; Treatment 1 (T1) was a diet with 1.5g/kg each of ammonium formate and calcium propionate and Treatment 2 (T2) a diet with 1g/kg each of calcium propionate, ammonium formate and calcium lactate. The control treatment included a starter diet (0 to 3 weeks) and a finisher diet (4 to 6 weeks). After thorough mixing of ingredients, the acid salts, which were in powder form, were mixed at the stated concentrations. The starter and finisher diets were formulated to meet or exceed the nutrient requirements as per the Bureau of Indian Standard (BIS1992). Ingredient and chemical composition of the basal diets are presented in Table 1. 


Table 1.  Ingredient and chemical composition of broiler starter and finisher diets

 

Starter

Finisher

Ingredients, air dry basis, g/kg

 

Ground maize

530.5

481.5

Soybean meal

400

320

De-oiled rice bran               

30

119.6

Di-calcium phosphate

18

18

Limestone powder

12

12

Soyabean oil       

-

40

Trace mineral mixa              

1

1

Iodized salt

3

2.9

Ventrimixb

0.4

0.4

Ventribee plusc

0.5

0.5

Choline chloride (60%)

1

1

Furazoidone        

0.2

0.2

Livoliv

0.5

0.5

Maduramycin

0.5

0.5

Vitamin E (50%)

0.1

0.1

L-lysine

0.5

0.3

DL-methionine

1.8

1.5

Acidifier

3

3

Chemical Composition

 

 

Dry Matter, g/kg

894.5

907.4

ME, KCal/kg+

2803

2920

Crude protein, g/kg            

217.2

192.1

Ether extract, g/kg

28.8

67.9

Crude fibre, g/kg

41.1

46.2

Total Ash, g/kg

84.2

84.4

Methionine, g/kg

5.4

4.8

Lysine, g/kg

12.2

1.02

aProvided per kg of diet: Zn, 60mg; Mn, 90mg; Fe, 110mg; KI, 2.5mg.

bEach g contains: Vitamin A-82, 500IU; Vitamin B2-50mg; Vitamin D3-12,000IU, Vitamin K-10mg.

cEach g contains: Vitamin B1-4mg; Vitamin B6-8mg; Vitamin B12-40mg; Vitamin E-40mg; Calcium D Pantothenate-40mg; Niacin-60mg


Housing and management

 

Chicks were housed in  battery type California cages which were cleaned thoroughly with formaldehyde and potassium permanganate solution (20 g KMnO4 + 40 ml formaldehyde solution per 100 square feet) three days prior to arrival of the birds. The day old chicks were offered an electrolyte solution upon arrival. The birds were maintained on a 24 hours constant light schedule. The brooding temperature was maintained close to their requirement, first by a heating device for the first three days.  The birds were vaccinated against Ranikhet disease and Infectious Bursal Disease on day 7, 14 and 21. Body weight was recorded before offering feed on the initial day, and then at weekly intervals up to 6 weeks. Mortality was recorded as it occurred. Body weight gain and feed intake were obtained by calculation. Feed conversion ratio (FCR) and feed intake were recorded at weekly intervals.  

 

Chemical analysis of feed samples

 

The dietary ingredients were analyzed for dry matter (DM), total ash and organic matter (OM), crude fibre (CF), ether extract (EE), nitrogen free extract (NFE) as per AOAC (1995).

 

Measurement of pH in different parts of the gastrointestinal tract

 

To determine the pH, 10 g of gut content from crop, proventriculus, gizzard, duodenum, jejunum and ileum were collected aseptically in 90 ml sterilized physiological saline (1: 10 dilution) (Al-Natour and Alshawabkeh 2005) and pH was determined.

 

Measurement of villus height

 

For measurement of villus height, parts of the small intestine were collected during slaughter. Paraffin sections were made from formalin-fixed tissue samples and then were stained with Delafield’s hematoxyline and eosin and mounted on Distrene Plasticiser Xylene. Heights of intestinal villus were measured by ocular micrometer under 10x objective of microscope. The reading was taken from ocular micrometer and the actual villus height was obtained by multiplying the ocular micrometer reading by conversion factor derived from stage micrometer (Lillie 1965). The heights of intestinal villus were expressed as micrometers (m).

 

Microbial count in feed and intestinal content

 

In order to count coliforms, Escherichia coli, Salmonella and fungal loads, 1 g of feed sample was taken in a sterile test tube and 10 ml of 0.9% sodium chloride solution was added to the test tube. Then the solution was mixed on a cyclomixer. 100 μl supernatants were taken and charged to Mac-conkey agar, E.M.B. agar, Xylose Lysine De-oxycholate Agar Plate and Sabouraud dextrose agar for counting coliforms, Escherichia coli, Salmonella and fungi, respectively (Garrido et al 2004: Andrews 1992). These were then incubated for 24 hrs at 370C. After incubation coliform was pink in colour, Escherichia coli had a metallic sheen, and Salmonella and fungal colonies were observed:  Aspergillus fumigatus was green, Aspergillus flavus was yellow and Aspergillus niger was black in colour. For counting Clostridium perfringens, 1 g of feed sample was taken in a sterile test tube and 10 ml of 0.9% NaCl solution was added and mixed in a cyclomixer. Supernatant (500 μl) was mixed to plates containing perfringens agar media,

which was then cooled at 450C (Garrido et al 2004: Andrews1992).  

 

Bacterial count in intestinal contents

 

Two birds from each replicate were electrically stunned and sacrificed at 6 weeks of age. Intestines,  including duodenum, jejunum, ileum and caecum were removed and ligated at both sides. Then tissue samples were placed in 50 ml tubes in sterile saline (0.9 g sodium chloride in 100 ml distilled water) and then kept at 40C until used for intestinal sampling. Serial dilutions of collected samples from different parts of intestinal contents were made up to the fifth dilution with sterile saline (0.9 g sodium chloride in 100 ml distilled water) and different bacterial loads of the gut contents were enumerated by the pour plate method (Quinn et al 1992).

 

Statistical analysis

 

Data were analysed using the General Linear Model of SPSS (1997) with replicates as experiment units for studying the effect of organic acid on broiler performance and gut health. Levels of significance were calculated as per the standard method described by Duncan (1995) whenever any effect was found significant.
 

 

Results and discussion

 

Performance

 

In Table 2 it can be seen that there were no significant differences among treatments in body weight (BW) and body weight gain (BWG) both at day 21 and 42.


Table 2.  Effect of organic acid salts on the performance of broilers

 

Virginiamycin,
0.5 g/kg

Ammonium formate + calcium propionate, 1.5g/kg of each

Ammonium formate + calcium propionate + calcium lactate, 1g/kg of each

SEM

P Value

Body weight, g

 

 

 

D 0

39.9

39.7

41.2

0.56

0.455

D 21

546

554

559

3.48

0.461

D 42

2035

1989

2090

21.28

0.370

Body weight gain, g

 

 

 

D 0-21

506

514

517

3.22

0.129

D 0-42

1995

1949

2049

21.08

0.353

Feed intake, g

 

 

 

D 0-21

828a

793b

789b

2.89

0.032

D 0-42

3495a

3182b

3380b

15.43

0.021

Feed conversion ratio, g/g

 

 

 

D 0-21

1.63a

1.52b

1.52b

0.004

0.011

D 0-42

1.75a

1.63b

1.65b

0.010

0.055

 a-cMeans within row with no common superscripts differ significantly (P<0.05)


Cumulative feed intakes after the 3rd and 6th weeks were higher (P<0.05) in the antibiotic group compared with the acidifier treated groups. It was also seen that feed conversion ratio after the 3rd and 6th week was better (P<0.05) in the acidifier treatments compared with the antibiotic treated group. Though the BWG of birds were similar between treatments, acidifier supplementation reduced feed intake which improved the FCR of acidifier treated birds. This finding corroborates with the observation of Pinchasov and Jensen (1989) where they studied several organic acids, including formic and propionic acid, and showed that only propionic acid had a significant action in reducing feed intake. Cave (1978) indicated that propionic acid plays a role in the satiation regulatory system, since intraperitoneal injection of the organic acid in broilers suspended intake for 0.5 to 1.5 hrs. Veeramani et al (2003) and Samanta and Biswas (1995) reported increased body weight with a lactic acid additive compared with birds fed without an antibiotic and acidifier. Hadorn et al (2000) and Partanen et al (2002) also reported that an acid combination intensified response compared with individual acids given alone. Better feed conversion ratio was also reported by Vogt et al (1982) and Veeramani et al (2003) in broilers given diets with propionic acid.

 

Effect on feed microbes

 

Feed sample analysis showed no significant difference in coliform and clostridium counts between the antibiotic and acidifier treated groups (Table 3).


Table 3.  Effect of organic acid salts on feed and intestinal microbial content of broilers

 

Virginiamycin,
0.5 g/kg

Formate + propionate, 1.5g/kg each

Formate + propionate
 + lactate, 1g/kg each

SEM

P Value

 Feed sample, CFU/gm of feed  sample

Coliforms (102)

11.5ab

13.5a

10.5b

2.887

0.037

Escherichia coli(102)

-

1.5

-

-

-

Salmonella

-

-

-

-

-

Clostridium sp.

110

135

135

3.33

0.118

Aspegillus  fumigatus (102)

2

-

-

-

Aspergillus flavus (102)

2

1

-

 -

-

Aspergillus niger (102)

-

-

-

 -

-

 Intestinal content, CFU/gm of intestinal content

Escherichia coli (105)

9.5

7.5

1.5

0.75

0.104

Salmonella

-

-

-

-

-

Clostridium sp. (105)

2.5

4

3.8

0.33

0.286

a-bMeans within row with no common superscripts differ significantly (P<0.05)


Escherichia coli was found in the formate + propionate supplemented group. Aspergillus fumigatus and Aspergillus flavus were present in higher amounts in the antibiotic treated group compared with the acidifier treated groups. This shows that the organic acid salt combination was as effective as the antibiotic in reducing coliform and clostridium counts in feed.  Singh and Verma (1970), Reusse (1977), Van Staden et al (1980), Sebastian et al (1996) and Blanchard et al (2001) also reported a decrease in Salmonella, Escherichia coli and mould counts in feeds with propionic and lactic added at different levels in broiler diets. The extra beneficial effect of the acidifiers, particularly propionic acid or propionate, was that they were more effective in reducing mould in feed in comparison to antibiotics (Dhawale 2005).

 

Effect on gut microbes

 

Though the total viable number of Escherichia coli and clostridium in gut contents varied numerically among treatments, the values were statistically non-significant (Table 3). This result indicates that the acidifier combination was as effective as the antibiotic in reducing Escherichia coli and clostridium counts in gut content. Thus the acidifier was shown to have both anti-mould and antibacterial properties. A similar observation was also recorded by Hinton and Linton (1988), Izat et al (1990b), Oliveira (1996) and Byrd et al (2001), who reported that the addition of lactic acid, formic acid and propionic acid to the diet and water effectively reduced Escherichia coli, coliforms and  Salmonella in poultry.

 

Effect on villus height

 

From the result it was observed that duodenal villus height was highest (P<0.05) in formate+propionate+lactate supplemented group (table 4). But no significant difference was found in duodenal villus height between antibiotic and formate+propionate supplemented group.


Table 4.  Effect of organic acid salts on the villus height of small intestine and pH of the GI tract of broiler chickens at 42 days

 

Virginiamycin,
0.5 g/kg

 Formate + propionate, 1.5g/kg each

Formate + propionate
 + lactate, 1g/kg each

SEM

P Value

Villus height, m

 

 

 

 

 

Duodenum (x10-3)

1158b

1119b

1456a

15.5

0.025

Jejunum (x10-3)

1017b

1124a

1029b

8.50

0.049

Ileum (x10-3)

547c

607b

672a

6.75

0.036

pH of GI tract

 

 

 

 

 

Crop

5.03

4.78

4.90

0.04

0.167

Proventriculus

2.43

3.10

3.15

0.06

0.051

Gizzard

3.03

2.25

3.35

0.19

0.700

Duodenum

6.25

6.10

6.30

0.15

0.854

Jejunum

6.65

6.30

6.95

0.25

0.368

Ileum

7.55

7.45

7.50

0.03

0.510

a-cMeans within row with no common superscripts differ significantly (P<0.05)


Jejunal villus height was higher (P<0.05) in formate+propionate supplemented group. Highest villus height of ileum was found highest in formate+propionate+lactate supplemented birds. This result revealed that combination of supplemental acidifier improved or maintained similar villus height when compared with antibiotic supplementation. Earlier workers [Pelicano et al (2005); Loddi et al (2004)] also noted higher villus height in duodenum and jejunum with most organic acidifiers added to broiler diets. The increase of villus height of the different segments of the small intestine may be attributed to the intestinal epithelium acting as a natural barrier against pathogenic bacteria and toxic substances that are present in the intestinal lumen. Stressors, pathogens and chemical substances, among others, cause disturbances in the normal microflora or in the intestinal epithelium that may alter the permeability of this natural barrier, facilitating the invasion of pathogens and prejudicial substances, modifying the metabolism, the ability to digest and absorb nutrients, leading to chronic inflammatory processes at the intestinal mucosa (Hofstad 1972; Podolsky 1993). So organic acidifiers reduce the growth of many pathogenic or non-pathogenic intestinal bacteria, therefore, reduce intestinal colonization and reduce infectious processes, ultimately decrease inflammatory processes at the intestinal mucosa, which increase villus height and function of secretion, digestion and absorption of nutrients can be appropriately performed by the mucosa (Iji and Tivey 1998; Pelicano et al 2005; Loddi et al 2004).

 

Effect on pH of different segments of the GI tract

 

No significant difference was found in pH of different segments of the G.I. tract due to supplementation of different organic acid salts compared with the antibiotic treated group (Table 4). These findings are consistent with the observation of  Izat et al (1990a), Mathew et al (1991) and Hernandez et al (2006) who reported no effect on intestinal pH with the use of Luprosil-NC (a product containing 53.5% propionic acid) and formic acid. This is because of the strong buffering action of the GI tract in poultry.

 

In conclusion, birds given the combination of acidifiers showed lower feed intake but similar body weight gain after 6t weeks compared with antibiotic-treated birds. Acidifier treatment also improved FCR. Acidifier treatment reduced bacterial counts to the same extent as the antibiotic treated group,  and also reduced fungal counts and improved villus height.
 

 

References

 

Al-Natour M Q and Alshawabkeh K M 2005 Using varying levels of formic acid to limit growth of Salmonella gallinarum in contaminated broiler feed. Asian Australian Journal of Animal Sciences 18: 390-395

 

Andrews W 1992 Manual of Food Quality Control. 4 Review 1. Microbiological analysis. Food and Agriculture Organization of United Nations, Rome

 

AOAC (Association of Official Analytical Chemists) 1995 Official method of the Association of Official Analytical Chemists.Volume 1, 16th edition. AOAC International, Arlington, USA

 

BIS 1992 Nutrient Requirements for poultry. IS: 13574: 1992

 

Blanchard P J, D’Mello J P F, Macdonald A M C, Catton S and Roser U 2001 Minimum inhibition concentrations for propionic acid and organic acid mixtures against storage fungi. World Mycotoxin Forum pp. 62 – 62

 

Byrd J A, Hargis B M, Caldwell D J, Bailey R H, Herron K L, McReynolds J L, Brewer R.L, Anderson R C, Bischoff K M, Callaway T R and Kubena L F 2001 Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter. Poultry Science 80: 278 – 83

 

Cave N A G 1978 The influence of non-sterified fatty acids on feeding activity   of chicks. Poultry Science 57: 1124-1128

 

Dhawale A 2005 Better eggshell quality with a gut acidifier. Poultry International. Volume 44: (4) 18-21

 

Duncan D B 1995 Multiple range and F-test. Biometrics 11:1 – 42

 

Garrido M N, Skjervheim M, Oppegaard H and Sorum H 2004 Acidified litter benefits the intestinal flora balance of broiler chicken. Applied Environmental Microbiology, 70: 5208-5213. 2004

 

Hadorn R H, Wiedmer and Feuerstein D 2000 Effect of different dosages of an organic acid mixture in broiler diets. Archive Geflugelk 65: 22 – 27

 

Hernandez F, Garcia V, Madrid J, Orengo J, Catala P and Megias M D 2006 Effect of formic acid on performance, digestibility, intestinal histomorphology and plasma metabolite levels of broiler chicken. British Poultry Science 47: 50 – 6 (7)

 

Hinton M and Linton A H 1988 Control of Salmonella infections in broiler chicken by the acid treatment of their feed. Veterinary Record 123: 416 – 421

 

Hofstad M S 1972 Editor diseases of poultry. 6th edition. Ames: The Iowa State University Press, 1176 p

 

Iji P A and Tivey D R 1998 Natural and synthetic oligosaccharide in broiler chicken diet. World Poultry Science Journal 54: 129 – 143

 

Izat A L, Adams M H, Cabel M C, Colberg M, Reiber M A, Skinner J T and Wealdroup P W 1990a. Effects of formic acid or calcium formate in feed on performance and microbiological characteristics of broilers. Poultry Science 69: 1876 – 1882

 

Izat A L, Tidwell N M, Thomas R A, Reiber MA, Adams M H, Colberg M and Waldroup P W 1990b Effects of a buffered propionic acid in diets on the performance of broiler chickens and on the microflora of the intestine and carcass. Poultry Science 69: 818 – 826

 

Lillie R D 1965 Histopathological technique and practical histochemistry. 3rd edn. Pp. 117 McGraw Hill Book Company, New York

 

Loddi M M, Maraes V M B, Nakaghi I S O, Tucci F, Hannas M I and Ariki J A 2004 Mannan oligosaccharide and organic acids on performance and intestinal morphometric characteristics of broiler chickens. In proceedings of the 20th annual symposium. Supplement. 1, p. 45

 

Mathew A G, Sutton A L, Scheidt A, Bforsyth D M, Patterson J A and Kelly D T 1991 Effect of a propionic acid containing feed additives on performance and intestinal microbial fermentation of ten weanling pig.In proceding of V th International symposium ondigestive physiology in pigs. Wageningen, Netherlands, Eaap Publication,No.54:pp.464-469

 

Nursey I 1997 Control of Salmonella. Kraftfutter 10: 415-22

 

Oliveira E O 1996 Uso de acidos graxos de cadeia no controle de salmonella emragoes de aves. Piracicaba. USP/ESALQ, p. 72 (Dissertagao-Mestrado)

 

Partanen K, Siljander-Rasi H, Alniuhkoln T, Suomi K and Possi M 2002 Performance of growing finishing pigs fed medium or high fibre diets supplemented with avilamycin, formic acid or formic sorbate blend. Livestock Production Science 73: 139 – 152

 

Pelicano E R L, Souza P A , Souza H B A, Figueiredo D F, Boiago M M, Carvalho S R and Bordon V F 2005 Intestinal mucosa development in broiler chicken fed natural growth promoters. Revista Brasileira de Ciencia Avicola,7 Campina http://www.scielo.br/pdf/rbca/v7n4/28744.pdf

 

Pinchasov Y and Jensen LS 1989 Effect of short chain fatty acids on voluntary feed intake of Broiler chicks. Poultry Science 68:1612-1618

 

Podolsky D K 1993 Regulation of intestinal epithelial proliferation: a few answers, many question. Animal Journal Physiologic 264: 179 – 186

 

Quinn P J Carter M E, Markey B K and Carter G R 1992 Clinical Veterinary Microbiology. Mosby-year book Europe limited Lynton House, 7-12 Tavistock square, London. pp. 61-65

 

Reusse U 1977 Possibility of salmonella decontamination of feeds with special reference to propionic acid – preliminary report. Dtsch tierarztl Wochenschr 84: 55–7

 

Russell J B and Diez-Gonzalez F 1998 The effects of fermentation acids on bacterial growth. Advance Microbiology and Physiology 39: 205-234

 

Samanta M and Biswas P 1995 Effect of feeding probiotic and lactic acid in the performance of broilers. Indian Journal of Poultry Science 30: 145 – 147

 

Sebastian S, Phillip L E, Fellner V, Idzian E S 1996. Comparative assessment of bacterial inoculation and propionic acid treatment of aerobic stability and microbial population of ensiled high moisture ear corn. Journal of Animal Science 74: 447–56  http://jas.fass.org/cgi/reprint/74/2/447

 

Singh-Verma S B 1970 Application of propionic acid for preserving mixed feeding stuffs, industrially prepared as well as cereals and maize. Results of microbiological studies. Zentralbl Bakteriol Parasitenkd Infektionskr Hygiene 125: 100

 

SPSS 1997 Base Application Guide 7.5 SPSS, USA

 

Van Staden J J, Vander Made H N and Jordaan E 1980 The control of bacterial contamination in carcass meal with propionic acid. Journal of Veterinary Research 47: 77 – 82

 

Veeramani P, Selvan S T and Viswanathan K 2003 Effect of acidic and alkaline drinking water on body weight gain and feed efficiency in commercial broiler. Indian Journal of Poultry Science 38: 42 – 44

 

Vogt H, Mathes S and Harnisch S 1982 The effect of organic acids on productivity of broilers. 2. Archiv-fur-Geflugelkunde 46: 223 – 227



Received 7 July 2007; Accepted 17 July 2007; Published 1 November 2007

Go to top