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CAN Technologies, Inc. Protocol

Effect of Levucell™ and Bactocell™ performance of broiler chickens challenged with necrotic enteritis

-Saccharomyces cereviseae (Levucell™ improved growth performance almost significantly in

chickens challenged with necrotic enteritis. Pediococcus acidilactici (Bactocell™) did not

improve performance or onset of lesions in chickens challenged with necrotic enteritis.

Three hundred and fifteen 1-d-old Ross 308 male broiler chickens were randomly

assigned to 5 dietary treatments, including an unchallenged positive control, a challenged

negative control without antibiotic, and a challenged positive control with virginiamycin (Vm).

Levucell and Bactocell were included in two separate treatments at 0.01% (100 g/ton). All

treatments, except the unchallenged positive control, were challenged with necrotic enteritis

(NE). Treatments contained 9 replications of 7 birds each. All birds received a common basal

diet based on corn-soybean meal and containing rye, fishmeal, and wheat midds. The diet was

then supplemented with the test products described in Table 1, which were added at the

expense of corn. On d 8, all birds except the unchallenged control, received a coccidia vaccine

(Cocci-vac™) via feed. On d 12, 13, 14, and 15, all birds, except the unchallenged positive

control group, received a Clostridium perfringens inoculum via feed. On d 14 and 15, a group of

challenged birds was euthanized to expose their digestive tract and evaluate the degree of

challenge that the birds had been exposed. Body weight gain (BWG), feed intake, and gain to

feed ratio (GF) were calculated at 7, 12, and 20 d of age. Lesion score was obtained from

selected treatments.

Statistical analysis Growth performance data was analyzed through ANOVA using the GLM procedure of SAS.

When considered appropriate, individual treatments were compared using orthogonal contrasts,

for which the P-value is reported.

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Table 1. Treatment description

Trt Description

1 Unchallenged Control

2 Challenged Control

3 As 2 + Virginiamycin

4 As 2 + Levucell™

5 As 2 + Bactocell™

Table 2. Additive Inclusion level and Active Component

Additive Inclusion Level Active Component Active Component

Inclusion Level Primary MOA

Levucell™ 0.01% Saccharomyces cereviseae 1 x 108

CFU/g Probiotic

Bactocell™ 0.01% Pediococcus acidilactici 2 E+6 CFU/g Probiotic

Table 3: Dietary Formulation

Ingredients Inclusion, %

Corn - Fine Ground 45.77

Wheat Midds By-Product 27-34% NDF 0.998

Soybean Meal - 48% Protein 29.360

SALT 0.287

CALCIUM CARBONATE 0.607

Oil - Corn 4.398

Phosphate - Mono Dicalcium 0.141

CHOLINE CHLORIDE-60 0.049

DL METHIONINE - DRY 0.179

L-LYSINE HCL 0.033

Fish Meal - HQ Menhaden 10.000

Vitamin Premix 0.119

POULTRY TRACE MINERAL 0.060

Rye 7.999

TOTAL 100.000

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Results -Birds receiving Levucell™ showed a trend indicating improvement in body weight at 20 d

growth performance with respect to birds from the negative control. No significant differences

were observed in body weight gain or gain to feed ratio.

-Birds receiving Bactocell™ did not show a significant improvement in live weight, body weight

gain, or gain to feed ratio.

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Table 4. Live weight (g/bird) and weight gain (g/bird) of broiler chickens challenged with necrotic enteritis and fed different direct-fed microbials

Live weight, g/bird Weight gain, g/bird

Trt 0 d 8 d 12 d 20 d 0-8 d 8-12 d 12-20 d 0-20 d

1 Unchall Ctrl 43.39 163.82c 344.56 775.46

c 121.74

c 184.04

ab 425.97

c 733.07

c

2 Chall Ctrl 43.80 172.14ab

358.63 807.63b 131.40

ab 184.10

ab 440.06

bc 764.21

b

3 Virginiamycin 43.83 172.49abc

362.22 852.10a 123.13

abc 195.07a 483.45

a 805.19

a

4 Levucell™ 43.33 173.73a 363.88 831.59

a 130.50

a 184.22

ab 462.45

ab 778.59

ab

5 Bactocell™ 43.20 168.52bc

352.78 813.69b 126.17

bc 175.68

b 455.22

bc 769.07

bc

SEM 0.74 3.39 7.51 10.06 3.40 5.82 13.33 14.58

CV 4.17 4.74 5.59 2.86 6.46 7.62 7.13 4.31

GLM P-values 0.9870 0.5470 0.4220 0.0090 0.2140 0.2540 0.0780 0.063

Orthogonal contrasts

Trt 3 vs Trt 2 -- 0.9270 0.3020 0.0070 0.0800 0.1800 0.0300 0.0590

Trt 4 vs Trt 2 -- 0.7560 0.8620 0.0610 0.8520 0.9880 0.2440 0.4760

Trt 5 vs Trt 2 -- 0.6390 0.3270 0.2130 0.2830 0.2840 0.3980 0.8090 a-e

Means with different superscript differ significantly (P < 0.05).

Table 5. Feed intake (g/bird) and gain to feed ratio (g:kg) of broiler chickens challenged with necrotic enteritis and fed different direct-fed microbials

Feed intake, g/bird Gain to feed ratio, g:kg

Trt 0-8 d 8-12 d 12-20 d 0-20 d 0-8 d 8-12 d 12-20 d 0-20 d

1 Unchall Ctrl 151.87 240.58b 596.74 998.17 808.70 776.69

a 717.55 741.12

2 Chall Ctrl 161.37 314.00a 551.25 1025.33 816.50 581.72

b 795.26 734.66

3 Virginiamycin 159.90 338.44a 596.02 1044.48 770.47 568.23

b 751.60 706.06

4 Levucell™ 166.98 308.96a 615.81 1091.14 784.51 606.39

b 758.91 733.08

5 Bactocell™ 158.35 319.54a 579.84 1058.9 800.71 586.63

b 796.75 726.13

SEM 5.46 14.84 24.89 21.516 22.68 33.54 22.68 21.52

CV 8.45 11.15 9.75 4.876 6.85 12.28 6.99 4.88

GLM P-values 0.5410 0.0040 0.3930 0.0670 0.6000 0.0020 0.0880 0.0670

Orthogonal contrasts

Trt 3 vs Trt 2 0.8430 0.2490 0.2110 0.5220 0.1390 0.7750 0.1640 0.1590

Trt 4 vs Trt 2 0.4790 0.8010 0.0680 0.0380 0.3240 0.5860 0.2550 0.9390

Trt 5 vs Trt 2 0.6830 0.7840 0.4030 0.2690 0.6230 0.9140 0.9620 0.6570 a-d

Means with different superscript differ significantly (P < 0.05).

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Figure 1. Weight gain (g/bird) and gain to feed ratio (g:kg) from 12 to 20 d of age of broiler birds receiving different direct-fed microbials from 0 to 20 d of age

Figure 2. Weight gain (g/bird) and gain to feed ratio (g:kg) from 0 to 20 d of age of broiler birds receiving different direct-fed microbials from 0 to 20 d of age.

Weight gain (12-20 d) of broilers challenged with

necrotic enteritis

426.0

440.1

483.5

462.5455.2

380.0

400.0

420.0

440.0

460.0

480.0

500.0

Unchall Ctrl Chall Ctrl Vm Levucell Bactocell

g/b

ird

Weight gain (0-20 d) of broilers challenged with

necrotic enteritis

733.1

764.2

805.2

778.6769.1

680.0

700.0

720.0

740.0

760.0

780.0

800.0

820.0

Unchall Ctrl Chall Ctrl Vm Levucell Bactocell

g/b

ird

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Figure 3. Gain to feed ratio (g:kg) from 0 to 20 d of age of broiler birds receiving different direct-fed microbials from 0 to 20 d of age

Gain to feed (0-20 d) of broilers challenged with

necrotic enteritis

741.12734.66

706.06

733.08726.13

680

690

700

710

720

730

740

750

Unchall Ctrl Chall Ctrl Vm Levucell Bactocell

g:k

g

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Table 8. Lesion score mean values for selected treatments

Lesion Score

Unchallenged + Ctrl 0.29d

Challenged Ctrl 1.25c

Vm 1.88a

Levucell™ 1.35bc

Bactocell™ 1.73ab

SEM 0.128

CV 23.217

GLM P-values 0.0000 a-d

Means with different superscript differ significantly (P < 0.05).