COMPANY WITH QUALITY SYSTEM CERTIFIED BY DNV

Via di S. Bartolo a Cintoia, 10450142 Firenze - ITALY

T +39 055 7332895F +39 055 780676

Email info@silospa.com www.silospa.com

SILO PRODUCTS ARE CERTIFIED UNDER

Chi

cken

sLi

ne

N NutritionProgram

Hea

lthPr

ogra

m

H

SILO patented 1-Monoglycerides from C1 to C7 for treating animals

Patent n. EP 2 410 871 B1

was established in 1950“ “104

The production area is about 30,000 square metres. The production capacity is 120,000 tons of Monoglycerides per year. SILO is the leader in the development and production of glycerides of short and medium chain fatty acids.

th:

• University of Parma - Professor Afro Quarantelli • University of Florence - Professor M. Antongiovanni • IRTA (Barcelona) • Universidad Autonoma de Barcelona - Professor Rafael Codony • Guelph University (Canada) - Professor Steve Leeson

-

-

Control: (no treatment) == == ==

Group 1: 104 P 0.5% 0.25% ==

Group 2: 104 P 0.5% 0.025% ==

Experimental groups - Treatments in feed 0-10 days 11-21 days 22-35 days

RESULTS

CONCLUSIONS

-

Evaluation of Eimeria tenella lesions in intestinal samples. Control Group 1 Group 2 Days

N° of chickens with lesions score 2 and 3 7 0 0 N° of chickens with lesions score 1 3 7 6 16N° of chickens with lesions score 0 0 3 4

N° of chickens with lesions score 2 and 3 7 0 0 N° of chickens with lesions score 1 3 1 6 21N° of chickens with lesions score 0 0 6 4 N° of chickens with lesions score 2 and 3 6 0 0 N° of chickens with lesions score 1 4 2 3 35N° of chickens with lesions score 0 0 8 7

CFU number of Clostridium perfringens in intestinal samples after 24 hours incubations at 37 °C

lortnoC Group 1

Group 2

CFU7000600050004000300020001000

0Days 16 21 35

Mortality: 2 chickens in the Control group died attributed to Eimeria and Clostridium lesions in the third period. No mortality was recorded in 104 P

0 = no lesions1 = limited petechial lesions 3 = hemorrhagic lesions

Scientific trial in isolators conducted by Public Animal HealthInstitute “Bruno Ubertini” - Forlì3 groups of Ross 308 SPF, 30 birds per group, were

placed in isolators at their first day of life. No

anticoccidial vaccination was implemented. All groups

were fed with the same commercial feed without

coccidiostats. Feed of the 2 experimental groups was

supplemented with in the first 21

days of life.

At day 5 of life each bird was infected via esophageal gavage

with 3000 sporulated oocysts of a mixed challenge of E.

acervulina, maxima and tenella and at day 10 -11 of life each

bird was challenged with 106 CFU of Clostridium perfringens.

At days 16-21-35 of life, 10 birds per group were sacrificed to

evaluate the intestinal lesions from coccidiosis and to quantify

CFU of Clostridium perfringens in intestinal samples.

groups.

2 = thickened walls with petechae bulges

104 P

104 P

Results demonstrated that even if administered at a dosage of 0.025% in feed, protects the entire intestine against Eimeria and Clostridium perfringes colonization.

CONTROL OF EIMERIA E CLOSTRIDIUM P.

In 2009 SILO patented the use of Monoglycerides from C1 to C7 with glycerol, as antibacterial and anti-mould substance for treating animals, under the International patent number EP2410871B1.

SILOhealth 104 is the result of numerous studies conducted in scientific collaboration with Guelph University (Prof. S. Leeson) and the Public Animal Health Institute “Bruno Ubertini” (Italy). SILOhealth 104 is a synergic combination of short, medium and long chain 1-Monoglycerides.

Furthermore SILOhealth 104 is the object of a second patent specific for its use in broiler chickens (patent n. 102016000082170)

SILOhealth 104 P can be used for poultry species in dry feed (pellets or mesh), to control dysbiosis and to increase the trophic action in the gut as follow:from the first day of life up to slaughtering: 0.08 - 0.2% in the feed

In order to achieve the increase of breast muscle weight up to forward reported values (see growth performance trial by Guelph University):

from the first day of life up to slaughtering: 0.35 - 0.5% in the feed

SILOhealth 104 L can be used for poultry species in dry feed (pellets or mesh), to control dysbiosis and to increase the trophic action in the gut as follow:from the first day of life up to slaughtering: 0.05 - 0.1% in the feed

In order to achieve the increase of breast muscle weight up to forward reported values (see growth performance trial by Guelph University):

from the first day of life up to slaughtering: 0.2 - 0.3% in the feed

104

Effects of SILOhealth 104 in broiler chickens, with a dose-response dosage starting from: 0.05%, in the feed:- stimulation of villi growth and tight junctions expression- reparation of lesions in gut mucosa caused by bacteria and intollerances- barrier effect against pathogens and intestinal integrity - dramatic increase of the intestinal surface for nutrients absorption- with a dosage of 0.2-0.3% of SILOhealth 104 in the feed you can get: increase of muscle mass of breast and thighs

The effects of Monobutyrin contained in SILOhealth 104 on villi were measured at University of Veterinary and Pharmaceutical Sciences - Brno - Institute of Pathological Morphology, by Prof. MVDr. Roman Halouzka. In the scientific trial, broiler chickens received 0.2% of product in drinking water during the first 15 days of life. The effects on villi growth were measured in different tracts of the gut and compared with a control group.

Central ileum

Control

+26.3%

Ileocaecal valve

Control

+7.8%

Meckel diverticulus

Control

+35.8%

EFFECTS ON VILLI

Comment of Prof. Halouzka: “The results show, and you can see it clearly, that the height of villi in some chicken groups increases towards the large intestine, which is surprising”

104

104

104

Trial design:

60 SPF birds were allotted to two groups and placed into isolators (30 birds per group). All the birds were orally infected with 107 CFU of Salmonella Typhimurium at the 7th day of age. The control group received a standard commercial feed; the treated group received the same feed supplemented with 0.3% of SILOhealth 104 L from day 1 to day 34 in the feed. 10 birds from each group were sacrificied at day 14, 24, 34; caecal samples were analized for CFU amount of Salmonella typhimurium after 24 hours incubation at 37°.

CFU OF SALMONELLA TYPHIMURIUMIN BIRDS CAECAL CONTENT AFTER INCUBATION

0

5000000

10000000

15000000

20000000

25000000

30000000

014

2434

Infection with

Control group

SILOhealth group

7days

CFU

CFU OF SALMONELLA TYPHIMURIUM IN CAECAL SAMPLES

Days Control group SILOhealth group

6.400.000 2.226.000Day 7 post-infection (14 days of life)

25.120.000 1.242.100

(high mortality) 300

Day 17 post-infection (24 days of life)

Day 27 post-infection (34 days of life)

Salmonella reduction in SILOhealth group = 6 log10

The inclusion of 0.3% of SILOhealth 104 L in feed reduced by 6 log10 the CFU amounts of Salmonella typhimurium in the birds caeca even in presence of high Salmonella typhimurium infection. This trial support the use of SILOhealth 104 L from early age to prevent Salmonella colonization.

TRIAL WITH BROILER CHICKENS EXPERIMENTALLY INFECTED WITH 107 CFU OF SALMONELLA TYPHIMURIUM

107 CFU of Salmonella t.

made at day 7th of life

-

It is known that free organic acids and their salts like Sodium and Calcium Butyrate have a very limited antibacterial efficacy at pH 6 or 7 (intestinal pH) since at neutral or slightly alcaline pH they are present in dissociated form. On the contrary, the 1-Monoglycerides contained in SILOhealth 104 do not dissociate and exert a strong antibacterial action against E. coli, Salmonella and Clostridium p.

The trials in vitro carried out by the Public Animal Health Institute “Bruno Ubertini” - Italy proved that the MIC (Minimum Inhibitory Concentration) of SILOhealth 104 L at pH 6-7 ranges between 0.06% e 0.12% against Salmonella, E-coli and Clostridium p., while benefitial Lactobacillus in not inhibited.

Regions where SILOhealth 104 is active

SILOhealth 104

IN VITRO STUDIES CONDUCTED BY THE PUBLIC ANIMAL HEALTH INSTITUTE “BRUNO UBERTINI”

RESULTS AFTER 35 DAYS

Experimental groups % of breast weight / final live weight

Control group 18.293a Treated group 1 19.980b

- MYOSTATIN

TREATMENTS

Control group240 ROSS 308

Group 1 treated with SILOhealth 104 L0.2% in the feed

104 L

+ BREAST

Treated group 2 20.065b

P < 0.05

GROWTHPERFORMANCE

Group2 treated with SILOhealth 104 L 0.3% in the feed

240 ROSS 308

After 5 weeks of age, the breast weight is significantly increased, compared to the final live weight, in the groups treated with 2000 and 3000 mg / kg of SILOhealth 104

Expression of both Forkhead box protein O4 (FOXO4) and myostatin, two factors that can inhibit protein synthesis, were found to be signifcantly decreased in the breast muscle of all SILOhealth 104 birds compared to control birds (P<0.05)

Study published on American Society of Animal Science, 2016: “Effects of butyrate glyceride product SILOhealth 104 L on the growth and performance of broiler chickens”Andrea Bedford*, Hai Yu*, Maria Hernandez*, E. James Squires**, Steve Leeson**, and Joshua Gong**Guelph Research and Development Centre, Agriculture and Agri-food Canada, Guelph, Ontario, Canada N1G 5C9** Department of Animal Bioscience, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Public Animal Health Institute “Bruno Ubertini” - L. Alborali, G. Tosi MIC on Salmonella spp., Clostridium p., E. coli spp.,

Lactobacillus acidophylus and plantarumof SILOhealth 104 compared to organic acids & sodium butyrate

pH Salmonella spp. Clostridium p. E. coli spp.

Lactobacillusacidophylus /

plantarum

SILOhealth 104 L 4.5 0.06% 0.01% 0.12% No inhibition

SILOhealth 104 L 7 0.06% 0,01% 0.12% No inhibition

Formic Acid 6 1.5% 1.8% 1.6% ==

Propionic Acid 6 2.4% 2.6% 2.5% ==

Sodium Butyrate 6 2.8% 3% 2.9% ==

-

It is known that free organic acids and their salts like Sodium and Calcium Butyrate have a very limited antibacterial efficacy at pH 6 or 7 (intestinal pH) since at neutral or slightly alcaline pH they are present in dissociated form. On the contrary, the 1-Monoglycerides contained in SILOhealth 104 do not dissociate and exert a strong antibacterial action against E. coli, Salmonella and Clostridium p.

The trials in vitro carried out by the Public Animal Health Institute “Bruno Ubertini” - Italy proved that the MIC (Minimum Inhibitory Concentration) of SILOhealth 104 L at pH 6-7 ranges between 0.06% e 0.12% against Salmonella, E-coli and Clostridium p., while benefitial Lactobacillus in not inhibited.

Regions where SILOhealth 104 is active

SILOhealth 104

IN VITRO STUDIES CONDUCTED BY THE PUBLIC ANIMAL HEALTH INSTITUTE “BRUNO UBERTINI”

RESULTS AFTER 35 DAYS

Experimental groups % of breast weight / final live weight

Control group 18.293a Treated group 1 19.980b

- MYOSTATIN

TREATMENTS

Control group240 ROSS 308

Group 1 treated with SILOhealth 104 L0.2% in the feed

104 L

+ BREAST

Treated group 2 20.065b

P < 0.05

GROWTHPERFORMANCE

Group2 treated with SILOhealth 104 L 0.3% in the feed

240 ROSS 308

After 5 weeks of age, the breast weight is significantly increased, compared to the final live weight, in the groups treated with 2000 and 3000 mg / kg of SILOhealth 104

Expression of both Forkhead box protein O4 (FOXO4) and myostatin, two factors that can inhibit protein synthesis, were found to be signifcantly decreased in the breast muscle of all SILOhealth 104 birds compared to control birds (P<0.05)

Study published on American Society of Animal Science, 2016: “Effects of butyrate glyceride product SILOhealth 104 L on the growth and performance of broiler chickens”Andrea Bedford*, Hai Yu*, Maria Hernandez*, E. James Squires**, Steve Leeson**, and Joshua Gong**Guelph Research and Development Centre, Agriculture and Agri-food Canada, Guelph, Ontario, Canada N1G 5C9** Department of Animal Bioscience, University of Guelph, Guelph, Ontario, Canada N1G 2W1

Public Animal Health Institute “Bruno Ubertini” - L. Alborali, G. Tosi MIC on Salmonella spp., Clostridium p., E. coli spp.,

Lactobacillus acidophylus and plantarumof SILOhealth 104 compared to organic acids & sodium butyrate

pH Salmonella spp. Clostridium p. E. coli spp.

Lactobacillusacidophylus /

plantarum

SILOhealth 104 L 4.5 0.06% 0.01% 0.12% No inhibition

SILOhealth 104 L 7 0.06% 0,01% 0.12% No inhibition

Formic Acid 6 1.5% 1.8% 1.6% ==

Propionic Acid 6 2.4% 2.6% 2.5% ==

Sodium Butyrate 6 2.8% 3% 2.9% ==

The effects of Monobutyrin contained in SILOhealth 104 on villi were measured at University of Veterinary and Pharmaceutical Sciences - Brno - Institute of Pathological Morphology, by Prof. MVDr. Roman Halouzka. In the scientific trial, broiler chickens received 0.2% of product in drinking water during the first 15 days of life. The effects on villi growth were measured in different tracts of the gut and compared with a control group.

Central ileum

Control

+26.3%

Ileocaecal valve

Control

+7.8%

Meckel diverticulus

Control

+35.8%

EFFECTS ON VILLI

Comment of Prof. Halouzka: “The results show, and you can see it clearly, that the height of villi in some chicken groups increases towards the large intestine, which is surprising”

104

104

104

Trial design:

60 SPF birds were allotted to two groups and placed into isolators (30 birds per group). All the birds were orally infected with 107 CFU of Salmonella Typhimurium at the 7th day of age. The control group received a standard commercial feed; the treated group received the same feed supplemented with 0.3% of SILOhealth 104 L from day 1 to day 34 in the feed. 10 birds from each group were sacrificied at day 14, 24, 34; caecal samples were analized for CFU amount of Salmonella typhimurium after 24 hours incubation at 37°.

CFU OF SALMONELLA TYPHIMURIUMIN BIRDS CAECAL CONTENT AFTER INCUBATION

0

5000000

10000000

15000000

20000000

25000000

30000000

014

2434

Infection with

Control group

SILOhealth group

7days

CFU

CFU OF SALMONELLA TYPHIMURIUM IN CAECAL SAMPLES

Days Control group SILOhealth group

6.400.000 2.226.000Day 7 post-infection (14 days of life)

25.120.000 1.242.100

(high mortality) 300

Day 17 post-infection (24 days of life)

Day 27 post-infection (34 days of life)

Salmonella reduction in SILOhealth group = 6 log10

The inclusion of 0.3% of SILOhealth 104 L in feed reduced by 6 log10 the CFU amounts of Salmonella typhimurium in the birds caeca even in presence of high Salmonella typhimurium infection. This trial support the use of SILOhealth 104 L from early age to prevent Salmonella colonization.

TRIAL WITH BROILER CHICKENS EXPERIMENTALLY INFECTED WITH 107 CFU OF SALMONELLA TYPHIMURIUM

107 CFU of Salmonella t.

made at day 7th of life

-

-

Control: (no treatment) == == ==

Group 1: 104 P 0.5% 0.25% ==

Group 2: 104 P 0.5% 0.025% ==

Experimental groups - Treatments in feed 0-10 days 11-21 days 22-35 days

RESULTS

CONCLUSIONS

-

Evaluation of Eimeria tenella lesions in intestinal samples. Control Group 1 Group 2 Days

N° of chickens with lesions score 2 and 3 7 0 0 N° of chickens with lesions score 1 3 7 6 16N° of chickens with lesions score 0 0 3 4

N° of chickens with lesions score 2 and 3 7 0 0 N° of chickens with lesions score 1 3 1 6 21N° of chickens with lesions score 0 0 6 4 N° of chickens with lesions score 2 and 3 6 0 0 N° of chickens with lesions score 1 4 2 3 35N° of chickens with lesions score 0 0 8 7

CFU number of Clostridium perfringens in intestinal samples after 24 hours incubations at 37 °C

lortnoC Group 1

Group 2

CFU7000600050004000300020001000

0Days 16 21 35

Mortality: 2 chickens in the Control group died attributed to Eimeria and Clostridium lesions in the third period. No mortality was recorded in 104 P

0 = no lesions1 = limited petechial lesions 3 = hemorrhagic lesions

Scientific trial in isolators conducted by Public Animal HealthInstitute “Bruno Ubertini” - Forlì3 groups of Ross 308 SPF, 30 birds per group, were

placed in isolators at their first day of life. No

anticoccidial vaccination was implemented. All groups

were fed with the same commercial feed without

coccidiostats. Feed of the 2 experimental groups was

supplemented with in the first 21

days of life.

At day 5 of life each bird was infected via esophageal gavage

with 3000 sporulated oocysts of a mixed challenge of E.

acervulina, maxima and tenella and at day 10 -11 of life each

bird was challenged with 106 CFU of Clostridium perfringens.

At days 16-21-35 of life, 10 birds per group were sacrificed to

evaluate the intestinal lesions from coccidiosis and to quantify

CFU of Clostridium perfringens in intestinal samples.

groups.

2 = thickened walls with petechae bulges

104 P

104 P

Results demonstrated that even if administered at a dosage of 0.025% in feed, protects the entire intestine against Eimeria and Clostridium perfringes colonization.

CONTROL OF EIMERIA E CLOSTRIDIUM P.

In 2009 SILO patented the use of Monoglycerides from C1 to C7 with glycerol, as antibacterial and anti-mould substance for treating animals, under the International patent number EP2410871B1.

SILOhealth 104 is the result of numerous studies conducted in scientific collaboration with Guelph University (Prof. S. Leeson) and the Public Animal Health Institute “Bruno Ubertini” (Italy). SILOhealth 104 is a synergic combination of short, medium and long chain 1-Monoglycerides.

Furthermore SILOhealth 104 is the object of a second patent specific for its use in broiler chickens (patent n. 102016000082170)

SILOhealth 104 P can be used for poultry species in dry feed (pellets or mesh), to control dysbiosis and to increase the trophic action in the gut as follow:from the first day of life up to slaughtering: 0.08 - 0.2% in the feed

In order to achieve the increase of breast muscle weight up to forward reported values (see growth performance trial by Guelph University):

from the first day of life up to slaughtering: 0.35 - 0.5% in the feed

SILOhealth 104 L can be used for poultry species in dry feed (pellets or mesh), to control dysbiosis and to increase the trophic action in the gut as follow:from the first day of life up to slaughtering: 0.05 - 0.1% in the feed

In order to achieve the increase of breast muscle weight up to forward reported values (see growth performance trial by Guelph University):

from the first day of life up to slaughtering: 0.2 - 0.3% in the feed

104

Effects of SILOhealth 104 in broiler chickens, with a dose-response dosage starting from: 0.05%, in the feed:- stimulation of villi growth and tight junctions expression- reparation of lesions in gut mucosa caused by bacteria and intollerances- barrier effect against pathogens and intestinal integrity - dramatic increase of the intestinal surface for nutrients absorption- with a dosage of 0.2-0.3% of SILOhealth 104 in the feed you can get: increase of muscle mass of breast and thighs

COMPANY WITH QUALITY SYSTEM CERTIFIED BY DNV

Via di S. Bartolo a Cintoia, 10450142 Firenze - ITALY

T +39 055 7332895F +39 055 780676

Email info@silospa.com www.silospa.com

SILO PRODUCTS ARE CERTIFIED UNDER

Chi

cken

sLi

ne

N NutritionProgram

Hea

lthPr

ogra

m

H

SILO patented 1-Monoglycerides from C1 to C7 for treating animals

Patent n. EP 2 410 871 B1

was established in 1950“ “104

The production area is about 30,000 square metres. The production capacity is 120,000 tons of Monoglycerides per year. SILO is the leader in the development and production of glycerides of short and medium chain fatty acids.

th:

• University of Parma - Professor Afro Quarantelli • University of Florence - Professor M. Antongiovanni • IRTA (Barcelona) • Universidad Autonoma de Barcelona - Professor Rafael Codony • Guelph University (Canada) - Professor Steve Leeson