Performance and nutrient utilization of broilers fed diets ... et al. - 2011.pdf · Performance and nutrient utilization of broilers ... T wo experiments were conducted to determine

DESCRIPTION OF PROBLEM

The increases in the cost of feed ingredients along with society’s growing expectations of

reduced environmental pollution from animal agriculture have been fueling interest in the use of enzymes in animal diets. Research on the use of exogenous enzymes in broiler diets has been

© 2011 Poultry Science Association, Inc.

Performance and nutrient utilization of broilers fed diets supplemented with a novel

mono-component protease

D. M. Freitas,* S. L. Vieira,*1 C. R. Angel,† A. Favero,* and A. Maiorka‡

*Departamento de Zootecnia, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 7712, Porto Alegre, Rio Grande do Sul, 91540-000, Brazil; †Department

of Animal and Avian Sciences, University of Maryland, College Park 20742; and ‡Departmento de Zootecnia, Universidade Federal do Paraná, Curitiba, Paraná, Brazil

Primary Audience: Nutritionists

SUMMARY

Two experiments were conducted to determine the effect of adding an exogenous protease to corn-, soybean meal-, and meat and bone meal-based broiler diets. In the first experiment, 1,764 male Ross 308 broiler chicks were placed in 63 floor pens, with 28 birds per pen. There were 7 treatments, with 9 replicates each, fed in the starter (d 1 to 21) and grower (d 22 to 40) phases. The dietary treatments were a positive control, formulated with 3,050 and 3,150 kcal of ME/kg and 22.5 and 20% CP in the starter and grower phases, respectively, and a negative control, formulated with a 4.4% reduction in ME and CP as compared with the positive control diets. A mono-component protease (75,000 protease/g) was added to the negative control diets at 0, 100, 200, 400, 800, and 1,600 ppm of feed. Broilers fed the positive control diet grew better and had a better feed-to-gain ratio (FE) than did those fed the negative control diets, regardless of enzyme supplementation. Protease supplementation had no effect on BW; however, FE was improved in a quadratic manner as protease was increased. In experiment 2, a factorial arrange-ment of 2 protein (7% difference in CP), 2 energy (3% difference in ME), and 2 protease (0 and 200 ppm) concentrations was used, resulting in 8 treatments replicated 11 times (22 male Cobb 500 broilers per replicate). No 3-way interactions were observed for live performance measures. Broilers fed the high-protein and high-energy diets performed better (P ≤ 0.01) than those fed the low-protein and low-energy diets. Protease supplementation improved FE as well as digestibilities of fat and CP (P ≤ 0.01), regardless of dietary protein or energy concentration. The protease used in these studies improved FE and dietary determined AME values as well as dietary CP and fat digestibility values.

Key words: amino acid, broiler, protease, protein

2011 J. Appl. Poult. Res. 20 :322–334 doi: 10.3382/japr.2010-00295

1 Corresponding author: [email protected]

323FREITAS ET AL.: PROTEASE FOR BROILERS

ongoing for decades; however, commercial use of enzymes is more recent. The practical use of phytase in chicken diets is well established, and interest in research on and use of enzymes tar-geting other substrates has increased.

Most commercial enzyme products currently available have more than one enzyme activity, whereas fewer products have only one substrate specificity. Enzyme blends are products having more than one enzyme and are either combina-tions of mono-component enzymes, generated by mixing enzymes targeting defined feed sub-strate matrixes, or fermentation products from wild-type strains of microorganisms expressing a broad spectrum of enzyme activities. The lat-ter type of product has a combination of useful and nonuseful enzymatic activities side by side. The majority of the currently available enzyme blends target cell wall substrates and a reduction in digesta viscosity. These products have vary-ing concentrations of xylanases, β-glucanases, and cellulases, but also have other enzyme ac-tivities, such as amylases, proteases, and lipases. In contrast, mono-component enzyme products, in general, have one organism of origin and are produced by selecting the DNA coding for the enzyme protein, which is then cloned into a host microbial strain. During fermentation, the microorganism with the cloned DNA releases the desired enzyme into the fermentation broth, which is then further purified.

Both the diversity of feed ingredients used in broiler diets and the variability within these ingredients affect nutrient digestibility. Protein digestibility, for instance, is high in typical corn- and soybean meal-based broiler feeds but tends to be lower in those containing animal-derived meals. The lower digestibility of these animal-derived meals may be due to excessive thermal processing, but also to the type of animal by-products it contains [1–7]. Variation in the di-gestibility of protein and amino acids (AA) also occurs within the same feed ingredient [1, 4, 8]. A wide range of endogenous proteases are syn-thesized and released in the gastrointestinal tract of the bird, and these are generally considered sufficient to optimize feed protein utilization [9, 10]. However, based on protein digestibility values reported in the literature, it appears that valuable amounts of protein pass through the

gastrointestinal tract without being completely digested [8]. This undigested protein presents an opportunity for the addition of specific exog-enous proteases.

The interpretation of results from studies conducted with proteases is sometimes difficult because of confounding effects of the presence of more than one enzymatic activity in a single treatment. Inconsistent and variable results are also frequently found because of feed ingredi-ent diversity and types of proteases that are often not clearly defined, as well as because of methodology differences [11–13]. On the other hand, research done with products with only one protease activity allows for easier in-terpretation; however, literature on this type of study with chickens is scarce. Ghazi et al. [14] reported positive effects of using a single-com-ponent protease, both in vitro and in vivo, when diets were deficient or marginal in AA. In other studies, large differences in bird responses were found between protease sources in terms of true N digestibility, but also on ME [15]. It has been hypothesized that the apparent improvements in the digestibilities of DM and energy found with supplemental proteases may result, in part, from negative feedback leading to a reduction in the endogenous production of proteases [16].

The objective of these studies was to evaluate the effects of a mono-component commercial serine protease added to broiler feeds at differ-ent concentrations on live performance, process-ing yields, and apparent ileal AA digestibility and apparent energy utilization.

MATERIALS AND METHODS

Two floor pen broiler chicken experiments (experiment) were conducted to evaluate live performance, processing yields, and apparent digestibilities of CP, fat, and energy as well as the AME of diets supplemented with an exog-enous protease. The enzyme used was a protease produced by submerged fermentation of Bacil-lus licheniformis containing transcribed genes from Nocardiopsis prasina. The enzyme activity for this protease is measured in protease units, with 1 unit defined as the amount of enzyme that releases 1 µmol of p-nitroaniline from 1 µM substrate (Suc-Ala-Ala-Pro-Phe-N-succinyl-

324 JAPR: Research Report

Ala-Ala-Pro-Phe-p-nitroanilide) per minute at pH 9.0 and 37°C. The branded product consists of 75,000 protease units/g of enzyme [17].

In both studies, lighting was continuous from placement to 14 d, after which a 12L:12D sched-ule was followed. Birds had free access to mash feed and water.

Amino acid analyses were conducted for all feed ingredients before feed formulation by us-ing HPLC-based methodology [18, 19]. Birds were culled if they could not walk properly or

when sex errors became evident. Mortality was recorded daily. Both studies were conducted in accordance with the Ethics and Research Com-mittees of the Universidade Federal do Rio Grande do Sul and the Universidade Federal do Parana, Brazil.

Experiment 1

Husbandry Practices. A total of 1,764 male Ross 308 broiler chicks vaccinated for Marek’s

Table 1. Broiler diets having graded increases in protease, experiment 11

Item

1 to 21 d 22 to 40 d

Positive control

Negative control

Positive control

Negative control

Ingredient, % Corn 56.34 62.13 62.64 68.71 Soybean meal 32.85 29.49 26.45 23.56 Meat and bone meal 6.20 6.70 5.98 6.03 Soybean oil 3.08 0.16 3.54 0.34 Limestone 0.52 0.52 0.49 0.49 Common salt 0.38 0.33 0.36 0.36 Na bicarbonate 0.09 0.13 — — dl-Methionine 0.19 0.11 0.15 0.06 l-Lysine-HCl 0.09 — 0.12 0.01 Vitamin and mineral mix2 0.22 0.22 0.22 0.22 Choline chloride 0.04 0.05 0.05 0.06 Kaolin (inert filler)3 — 0.16 — 0.16Calculated composition, % ME, kcal/kg 3,050 2,915 3,150 3,000 CP 22.5 21.5 20.0 19.0 Ca 1.00 1.00 0.95 0.95 Available P 0.50 0.50 0.47 0.47 Na 0.23 0.23 0.20 0.20 Total lysine 1.40 1.23 1.23 1.07 TSAA 0.98 0.87 0.87 0.75 Total threonine 0.99 0.96 0.88 0.84 Digestible lysine 1.27 1.12 1.12 0.97 Digestible TSAA 0.89 0.79 0.79 0.68 Digestible threonine 0.86 0.83 0.76 0.73Analyzed composition, % CP 23.2 22.6 21.6 19.9 Lysine 1.42 1.24 1.21 1.03 TSAA 1.01 0.89 0.86 0.76 Threonine 0.98 0.95 0.82 0.791Treatments: positive control and negative control. Crude protein and ME were reduced in the negative control by 4.4%, and digestible lysine and TSAA were reduced by 11.8 and 12.2%, respectively, as compared with the positive control diet.2Vitamin, mineral, and additive contributions per kilogram of feed: vitamin A, 9,000 IU; vitamin D3, 2,500 IU; vitamin E, 20 IU; vitamin K3, 2.5 mg; vitamin B1, 1.5 mg; vitamin B2, 6 mg; vitamin B6, 3 mg; pantothenic acid, 1.2 mg; biotin, 0.06 mg; folic acid, 0.8 mg; niacin, 25 mg; vitamin B12, 12 μg; I, 2 mg; Se, 0.25 mg; Cu, 20 mg; Mn, 160 mg; Zn, 100 mg; Fe, 100 mg (all sources as sulfate, except for sodium selenite and potassium iodate); nicarbazine, 98 ppm (1 to 21 d); salinomycin, 66 ppm (22 to 40 d); bacitracin methylene disalicylate, 55 ppm (1 to 40 d).3The protease replaced kaolin. Diet levels (protease units/kg of feed) were calculated (analyzed) for 1 to 21 d and 22 to 40 d, respectively: positive control, 0 to 0 (below detection limits); negative control, 0 to 0 (below detection limits); 7,500 (8,735 to 7,630); 15,000 (16,210 to 15,125); 30,000 (32,586 to 29,978); 60,000 (66,444 to 62,432); 120,000 (135,964 to 125,497).


disease were obtained from a commercial hatch-ery [20]. Broilers were placed in 63 pens, 28 in each (1.70 × 1.65 m), with 10 birds/m2. Shavings that had previously been used once were used as bedding, and each pen was equipped with 1 tube feeder (15-kg capacity) and 3 nipple drinkers.

Body weight, BW gain (BWG) feed intake (FI), and the feed-to-gain ratio (FE), corrected for the weight of dead birds, were determined weekly on a pen basis until d 40. After the final weighing, 8 birds were randomly marked with a plastic leg band and maintained on the same experimental feeds until processing on the next day. Birds were fasted for 8 h before slaugh-ter, individually weighed, and then electrically stunned, exsanguinated (bled for 3 min after a jugular vein cut), scalded at 60°C, mechanically feather picked, and manually eviscerated. Car-casses were chilled in ice water for 3 h and hung for 3 min to remove excess water, and yield mea-surements were taken. Abdominal fat removal and carcass cuts were performed by skilled in-dustry personnel to obtain deboned breast meat (pectoralis major + pectoralis minor) and bone-in thighs, drumsticks, wings, and cages. Carcass weights were expressed as a proportion of BW, whereas abdominal fat and carcass parts were expressed relative to the whole carcass.

Dietary Treatments. Corn, soybean meal, and meat and bone meal were used as the major ingredients to formulate diets in a 2-phase feed-ing program (starter from 1 to 21 d and grower from 22 to 40 d; Table 1). A positive control diet, formulated at or above the nutrient recom-mendations proposed by Rostagno et al. [21], and a negative control diet with CP and ME re-duced by 4.4% and digestible lysine and TSAA reduced by 11.8 and 12.2%, respectively, as compared with the positive control diets, were formulated. Five other dietary treatments having the same nutrient profile as the negative control had the protease added at graded concentrations (0, 100, 200, 400, 800, and 1,600 ppm), replac-ing the inert filler (kaolin). The reductions in CP, ME, digestible lysine, and TSAA were chosen to allow for a measurable response to the prote-ase. Diets were analyzed for CP, Ca, P, AA, and protease [22].

Statistical Analysis. A complete randomized block design with 7 treatments and 9 replicate pens per treatment was used. Analysis of vari-

ance was conducted using the GLM procedure of SAS [23]. When the effects were found to be significant, treatment means were separated us-ing Tukey’s honestly significant difference test [24]. Significance was accepted at P ≤ 0.05. Re-gression analysis was conducted only on nega-tive control diets to determine the effects of the graded concentrations of protease by using the REG procedure of SAS [23]. Carcass and mor-tality data were arcsine transformed before sta-tistical analysis.

Experiment 2

Husbandry Practices. A total of 1,936 male Cobb 500 male broiler chicks vaccinated for Marek’s disease were obtained from a commer-cial hatchery [25]. Broilers were placed in 88 pens (1.50 × 1.50 m), with 22 birds per pen and 9.8 birds/m2. Shavings that had previously been used once were used as bedding, and each pen was equipped with 1 tube feeder (18-kg capac-ity) and 1 bell drinker.

Live performance measurements (BWG, FI, and FE) were determined at each phase-feeding change as well as at the end of the 42-d trial. At 42 d, 5 birds per pen were randomly sampled from 8 randomly selected pens and killed by cervical dislocation, and the contents of the ile-um (from Meckel’s diverticulum to the ileocecal junction) were collected. The ileal samples were freeze-dried, ground to pass through a 3-mm sieve, and stored at −18°C until analysis. Ileal contents were analyzed for CP, AA, crude fat, and gross energy [22], and AME was calculated [26].

Dietary Treatments. Corn, soybean meal, and meat and bone meal were used as the major ingredients to formulate the feeds (Table 2) for a 4-phase program: 1 to 7 d, 8 to 21 d, 22 to 35 d, and 36 to 42 d of age, as defined in the Brazilian nutrient recommendation tables [21]. Dietary treatments resulted from a 2 × 2 × 2 factorial arrangement of 2 protein levels (high or low protein) with a 7% difference, 2 energy levels (high or low energy) with a 3% difference, and supplementation or no supplementation with protease at 200 ppm (15,000 protease units/kg of diet), following the manufacturer’s recom-mendations and based on guaranteed enzyme concentrations. The nutrient concentration dif-

326 JAPR: Research ReportTa

ble

2. B

roile

r die

ts w

ith h

igh

and

low

CP

and

ME

with

or w

ithou

t pro

teas

e, e

xper

imen

t 21

Item

1 to

7 d

8 to

21

d22

to 3

5 d

36 to

42

d2

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Ingr

edie

nt, %

Cor

n61

.57

56.2

859

.49

54.2

064

.98

60.0

062

.85

57.8

467

.71

63.1

265

.50

60.9

269

.48

65.1

667

.10

62.8

7 S

oybe

an m

eal

30.2

234

.50

30.6

034

.88

26.1

630

.12

26.5

230

.51

22.8

626

.60

23.2

627

.00

19.0

522

.52

19.4

722

.93

Mea

t and

b

one

mea

l6.

906.

806.

906.

807.

107.

107.

107.

106.

706.

606.

706.

606.

706.

706.

806.

70

Soy

bean

fat

0.05

0.90

1.78

2.62

0.63

1.42

2.42

3.21

1.55

2.30

3.38

4.12

2.64

3.33

4.52

5.21

Lim

esto

ne0.

130.

130.

120.

120.

070.

070.

060.

060.

160.

160.

150.

150.

160.

150.

150.

15 C

omm

on sa

lt0.

290.

260.

290.

260.

290.

260.

290.

260.

300.

280.

300.

280.

300.

280.

310.

29 N

a bi

carb

onat

e0.

060.

100.

050.

100.

050.

090.

050.

090.

040.

080.

040.

080.

040.

070.

030.

06 d

l-M

ethi

onin

e0.

250.

340.

250.

340.

210.

290.

210.

290.

170.

240.

170.

240.

130.

190.

130.

20 l

-Lys

ine

HC

l0.

190.

290.

180.

280.

180.

270.

170.

260.

160.

240.

150.

230.

150.

210.

140.

20 l

-Thr

eoni

ne0.

010.

070.

010.

07—

0.05

—0.

05—

0.03

—0.

03—

0.04

—0.

04 V

itam

in a

nd

min

eral

mix

30.

220.

220.

220.

220.

220.

220.

220.

220.

250.

250.

250.

250.

250.

250.

250.

25

Cho

line

chlo

ride

0.09

0.09

0.09

0.09

0.09

0.09

0.09

0.09

0.08

0.08

0.08

0.08

0.08

0.08

0.08

0.08

Kao

lin/p

rote

ase4

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

0 to

0.0

20

to 0

.02

ME,

kca

l/kg

2,88

52,

885

2,97

42,

974

2,96

02,

960

3,05

23,

052

3,05

03,

050

3,14

43,

144

3,15

03,

150

3,24

73,

247

Cal

cula

ted

c

ompo

sitio

n, %

CP

22.0

23.7

22.0

23.7

20.5

22.0

20.5

22.0

19.0

20.4

19.0

20.4

17.5

18.8

17.5

18.8

Ca

1.00

1.00

1.00

1.00

0.95

0.95

0.95

0.95

0.92

0.92

0.92

0.92

0.90

0.90

0.90

0.90

Ava

ilabl

e P

0.50

0.50

0.50

0.50

0.47

0.47

0.47

0.47

0.43

0.43

0.43

0.43

0.40

0.40

0.40

0.40

Na

0.23

0.23

0.23

0.23

0.20

0.20

0.20

0.20

0.18

0.18

0.18

0.18

0.17

0.17

0.17

0.17

Tot

al ly

sine

1.26

1.44

1.26

1.44

1.16

1.32

1.16

1.32

1.05

1.20

1.05

1.20

0.94

1.08

0.94

1.08

TSA

A0.

901.

030.

901.

030.

820.

940.

820.

940.

750.

850.

750.

850.

670.

760.

670.

76 T

otal

thre

onin

e0.

830.

940.

830.

940.

760.

860.

760.

860.

700.

780.

700.

780.

640.

730.

640.

73 D

iges

tible

lysi

ne1.

151.

321.

151.

321.

051.

211.

051.

210.

951.

090.

951.

090.

850.

980.

850.

98 D

iges

tible

TSA

A0.

820.

940.

820.

940.

750.

880.

750.

880.

670.

780.

670.

780.

600.

690.

600.

69 D

iges

tible

thre

onin

e0.

710.

820.

710.

820.

650.

750.

650.

750.

600.

680.

600.

680.

550.

630.

550.

63

Con

tinue

d


Tabl

e 2

(Con

tinue

d). B

roile

r die

ts w

ith h

igh

and

low

CP

and

ME

with

or w

ithou

t pro

teas

e, e

xper

imen

t 21

Item

1 to

7 d

8 to

21

d22

to 3

5 d

36 to

42

d2

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

ene

rgy

Hig

h en

ergy

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Low

pr

otei

nH

igh

pr

otei

nLo

w

prot

ein

Hig

h

prot

ein

Ana

lyze

d

com

posi

tion,

% C

P21

.122

.921

.322

.120

.021

.119

.821

.318

.719

.718

.919

.716

.818

.217

.217

.9 L

ysin

e1.

281.

431.

261.

431.

171.

351.

141.

351.

041.

231.

071.

230.

961.

100.

931.

09 T

SAA

0.94

1.01

0.91

1.05

0.84

0.97

0.88

0.97

0.78

0.86

0.77

0.87

0.69

0.87

0.69

0.80

Thr

eoni

ne0.

850.

950.

850.

970.

770.

880.

730.

850.

720.

790.

690.

790.

660.

730.

670.

74 P

rote

ase,

uni

ts/k

g515

,391

17,6

2614

,983

14,6

9415

,391

12,0

0617

,369

16,6

8515

,532

16,4

1813

,564

13,5

5613

,096

16,5

9617

,857

16,9

741 Tr

eatm

ents

: hig

h an

d lo

w C

P (h

igh

prot

ein

and

low

pro

tein

, with

a 7%

diff

eren

ce) a

nd h

igh

and

low

ME

(hig

h en

ergy

and

low

ener

gy, w

ith a

3% d

iffer

ence

), w

ith o

r with

out 2

00 p

pm o

f pro

teas

e (1

5,00

0 pr

otea

se u

nits

/kg)

.2 C

elite

(Cel

ite C

orpo

ratio

n, B

ogot

á, C

olom

bia)

was

add

ed o

n to

p at

1%

as a

mar

ker.

3 Vita

min

, min

eral

, and

add

itive

con

tribu

tions

per

kilo

gram

of f

eed:

vita

min

A, 9

,000

IU; v

itam

in D

3, 2,

500

IU; v

itam

in E

, 20

IU; v

itam

in K

3, 2.

5 m

g; v

itam

in B

1, 1.

5 m

g; v

itam

in B

2, 6

mg;

vi

tam

in B

6, 3

mg;

pan

toth

enic

aci

d, 1

.2 m

g; b

iotin

, 0.0

6 m

g; fo

lic a

cid,

0.8

mg;

nia

cin,

25

mg;

vita

min

B12

, 12

μg; I

, 2 m

g; S

e, 0

.25

mg;

Cu,

20

mg;

Mn,

160

mg;

Zn,

100

mg;

Fe,

100

mg

(all

sour

ces a

s sul

fate

, exc

ept f

or so

dium

sele

nite

and

cal

cium

ioda

te);

mon

ensi

n so

dium

, 100

ppm

(1 to

21

d); s

alin

omyc

in: 6

6 pp

m (2

2 to

to 4

0 d)

.4 Th

e pr

otea

se re

plac

ed k

aolin

.5 Pr

otea

se a

ctiv

ities

in d

iets

with

out a

dded

enz

yme

wer

e be

low

the

dete

ctio

n lim

its. T

he c

once

ntra

tions

pre

sent

ed a

re a

naly

zed

valu

es fo

r the

die

ts c

onta

inin

g pr

otea

se.


Tabl

e 3.

Liv

e pe

rform

ance

of b

roile

rs fe

d di

ets

with

gra

ded

conc

entra

tions

of p

rote

ase,

exp

erim

ent 1

1

Item

BW

G, g

Feed

inta

ke, g

FE

1 to

21

d21

to

40 d

1 to

40

d1

to

21 d

21 to

40

d1

to

40 d

1 to

21

d21

to

40 d

1 to

40

d

Trea

tmen

t2

Pos

itive

con

trol

896a

1,93

4a2,

831a

1,20

13,

348

4,55

01.

340a

1.73

1 ª

1.60

7a

Neg

ativ

e co

ntro

l82

1b1,

765b

2,58

1b1,

208

3,31

14,

522

1.47

0c1.

876b

1.75

2c

Neg

ativ

e co

ntro

l + 1

00 p

pm82

2b1,

755b

2,57

8b1,

202

3,31

24,

515

1.46

2c1.

888b

1.75

1c

Neg

ativ

e co

ntro

l + 2

00 p

pm83

7b1,

775b

2,60

5b1,

198

3,32

84,

526

1.43

1bc1.

875b

1.73

7bc

Neg

ativ

e co

ntro

l + 4

00 p

pm83

2b1,

764b

2,59

7b1,

192

3,31

94,

512

1.43

3bc1.

881b

1.73

7bc

Neg

ativ

e co

ntro

l + 8

00 p

pm82

9b1,

772b

2,60

1b1,

199

3,27

94,

478

1.44

6bc1.

851b

1.72

2bc

Neg

ativ

e co

ntro

l + 1

,600

ppm

839b

1,79

2b2,

631b

1,19

33,

315

4,50

91.

421b

1.85

0b1.

713b

SEM

7.08

14.5

217

.56

14.3

424

.32

63.6

70.

010

0.01

10.

006

P-va

lue

0.00

010.

0001

0.00

010.

990

0.63

60.

737

0.00

010.

0001

0.00

01a–

c Mea

ns w

ithin

a c

olum

n no

t sha

ring

a co

mm

on su

pers

crip

t diff

er (P

< 0

.05)

.1 M

eans

are

bas

ed o

n 9

repl

icat

e pe

ns p

er tr

eatm

ent.

2 Trea

tmen

ts: p

ositi

ve c

ontro

l and

neg

ativ

e co

ntro

l. C

rude

pro

tein

and

ME

wer

e re

duce

d in

the

nega

tive

cont

rol b

y 4.

4%, a

nd d

iges

tible

lysi

ne a

nd T

SAA

wer

e re

duce

d by

11.

8 an

d 12

.2%

, re

spec

tivel

y, a

s com

pare

d w

ith th

e po

sitiv

e co

ntro

l die

t.


ferences chosen for this experiment were based, in part, on the results from experiment 1 as well as on other work done with this protease [27]. Diets from 36 to 42 d had 1% Celite [28] added on top as a marker. Diets were analyzed for CP, crude fat, AA, gross energy, and protease [22].

Statistical Analysis. Live performance data were submitted to a 3-way ANOVA resulting from a 2 × 2 × 2 factorial arrangement with 11 replications per treatment, using the GLM pro-cedure of SAS [23]. For the digestibility data, there were 8 replications. Mortality data were arcsine transformed before statistical analysis. When the effects were found to be significant, treatment means were separated using Tukey’s honestly significant difference test [24]. Signifi-cance was accepted at P ≤ 0.05.

RESULTS

Experiment 1: Live Performance and Carcass Yield

In this study, a 2-phase feeding program was used for the purpose of determining whether there was a protease dose-response effect in a starter- and grower-phase feeding program. A positive control diet was formulated with nu-trient concentrations that met or exceeded the Brazilian nutrient recommendations [21]. Live performance results are shown in Table 3. Body weight gain and FE were improved in broilers

fed the positive control diets as compared with those fed the negative control diets, irrespective of the addition of protease. Protease supplemen-tation of the negative control diet had no effect on BWG, regardless of protease concentration; however, a quadratic improvement in FE was observed with supplementation of the negative control diets with protease from d 1 to 21 (y = 1.445 × 10−8 X2 – 4.699 × 10−5 + 1.461; P < 0.0092, R2 = 0.6813) and from 1 to 42 d (y = 1.345 × 10−8 X2 – 4.132 × 10−5 + 1.719; P < 0.0021, R2 = 0.2395). No effects were observed on FI, mortality, or carcass traits (Table 4). Mor-tality was not affected by treatment (overall mean = 1.8 ± 0.286%).

Experiment 2: Live Performance

Live performance results for experiment 2 are summarized in Table 5. Birds fed diets with high protein and high energy had improved BWG and FE when compared with those fed diets with low protein and low energy. Broil-ers fed the high-energy diets had greater BWG from d 1 to 21 and from d 1 to 42 as compared with those fed the low-energy diets. Enzyme supplementation had no effect on BWG, regard-less of the energy or protein concentration of the diet. However, adding the protease resulted in improvements in FE regardless of the dietary energy or protein. No 3-way interactions were observed. Mortality during the trial was not af-

Table 4. Carcass yield and commercial cuts from broilers fed diets with graded concentrations of protease (percentage at 41 d of age), experiment 11

Item BW, gCarcass yield, %

Abdominal fat, %

Deboned breast meat, %

Thighs, %

Wings, %

Drumsticks, %

Treatment2

Positive control 2,945a 78.4 2.79 26.6 18.6 10.3b 12.7 Negative control 2,754b 78.8 2.58 25.9 18.9 10.5ab 12.9 Negative control + 100 ppm 2,758b 78.4 2.33 25.7 18.7 10.8a 12.8 Negative control + 200 ppm 2,763b 78.4 2.60 25.6 18.7 10.3b 12.7 Negative control + 400 ppm 2,755b 78.6 2.33 26.0 18.6 10.6ab 12.9 Negative control + 800 ppm 2,756b 78.8 2.65 25.4 18.7 10.2b 12.7 Negative control + 1,600 ppm 2,761b 78.7 2.39 25.9 18.8 10.5ab 13.0SEM 10.69 0.30 0.21 0.26 0.22 0.12 0.14P-value 0.0001 0.867 0.377 0.22 0.989 0.0175 0.703a,bMeans within a column not sharing a common superscript differ significantly (P < 0.05).1Means were calculated from 8 birds taken randomly from each of 9 replicate pens per treatment. Carcass and parts are percent-age yields of the eviscerated carcass.2Treatments: positive control and negative control. Crude protein and ME were reduced in the negative control by 4.4%, and digestible lysine and TSAA were reduced by 11.8 and 12.2%, respectively, as compared with the positive control diet.

330 JAPR: Research ReportTa

ble

5. L

ive

perfo

rman

ce o

f bro

ilers

fed

diet

s w

ith 2

leve

ls o

f pro

tein

and

ene

rgy,

with

or w

ithou

t pro

teas

e, e

xper

imen

t 21

Item

BW

G, g

FI, g

FE

1 to

21

d22

to

42 d

1 to

42

d1

to

21 d

22 to

42

d1

to

42 d

1 to

21

d22

to

42 d

1 to

42

d

Trea

tmen

t2

Low

pro

tein

, low

ene

rgy,

no

prot

ease

735e

1,95

1b2,

686d

1,09

23,

627

4,71

21.

487a

1.86

0a1.

755a

Hig

h pr

otei

n, lo

w e

nerg

y, n

o pr

otea

se77

4bc1,

998ab

2,77

3abc

1,10

33,

580

4,67

91.

423c

1.79

2c1.

688b

Low

pro

tein

, hig

h en

ergy

, no

prot

ease

770cd

1,96

2ab2,

732bc

d1,

105

3,53

34,

636

1.43

5bc1.

802bc

1.69

7b

Hig

h pr

otei

n, h

igh

ener

gy, n

o pr

otea

se80

0ab2,

017ab

2,81

7ab1,

094

3,56

44,

649

1.36

7d1.

767cd

1.65

0cd

Low

pro

tein

, low

ene

rgy,

pro

teas

e74

3de1,

955b

2,69

8cd1,

087

3,60

84,

691

1.46

3ab1.

846ab

1.73

9a

Hig

h pr

otei

n, lo

w e

nerg

y, p

rote

ase

776bc

2,01

2ab2,

788ab

1,09

13,

569

4,65

61.

406c

1.77

4cd1.

670bc

Low

pro

tein

, hig

h en

ergy

, pro

teas

e77

1cd1,

973ab

2,74

4bcd

1,09

13,

534

4,61

91.

415c

1.79

1c1.

684b

Hig

h pr

otei

n, h

igh

ener

gy, p

rote

ase

809a

2,04

1a2,

850a

1,09

73,

557

4,64

61.

357d

1.74

4d1.

631d

SEM

6.77

18.4

219

.71

9.64

31.4

534

.53

0.01

20.

011

0.00

7M

ain

effe

ct P

rote

in

Low

755

1,96

02,

715

1,09

43,

576

4,66

41.

450

1.82

51.

718

H

igh

790

2,01

72,

807

1,09

63,

568

4,65

81.

389

1.76

91.

659

Ene

rgy

Lo

w75

71,

979

2,73

61,

093

3,59

64,

685

1.44

61.

818

1.71

3

Hig

h78

81,

998

2,78

61,

097

3,54

74,

638

1.39

41.

776

1.66

5 P

rote

ase

N

o77

01,

982

2,75

21,

099

3,57

64,

669

1.42

91.

805

1.69

7

Yes

775

1,99

52,

770

1,09

23,

567

4,65

31.

410

1.78

91.

681

P-va

lue

Sour

ce o

f var

iatio

n P

rote

in0.

0001

0.00

010.

0001

0.72

080.

7211

0.78

440.

0001

0.00

010.

0001

Ene

rgy

0.00

010.

1426

0.00

060.

6156

0.02

980.

0581

0.00

010.

0001

0.00

01 P

rote

ase

0.32

280.

3134

0.20

150.

2949

0.67

980.

2756

0.00

010.

0301

0.00

20 P

rote

in ×

ene

rgy

0.81

990.

7160

0.79

570.

4671

0.11

950.

2653

0.74

230.

0580

0.09

46 P

rote

in ×

pro

teas

e0.

9786

0.66

140.

6789

0.71

980.

9884

0.91

840.

5518

0.57

270.

7106

Ene

rgy

× pr

otea

se0.

9588

0.76

590.

8961

0.82

850.

7847

0.80

520.

5885

0.96

450.

9651

Pro

tein

× e

nerg

y ×

prot

ease

0.96

100.

9797

0.95

110.

3560

0.86

620.

8796

0.80

880.

7878

0.83

84a–

e Mea

ns w

ithin

a c

olum

n no

t sha

ring

a co

mm

on su

pers

crip

t diff

er (P

< 0

.05)

.1 M

eans

wer

e ba

sed

on 1

1 re

plic

ate

pens

per

trea

tmen

t.2 Tr

eatm

ents

: hig

h an

d lo

w C

P an

d M

E (d

iffer

ence

s of 7

and

3%

, res

pect

ivel

y), w

ith o

r with

out p

rote

ase

at 2

00 p

pm (1

5,00

0 pr

otea

se u

nits

/kg)

.


fected by dietary treatment (overall mean = 2.1 ± 0.378%).

Digestibility values for CP and fat, deter-mined at 42 d of age, were higher when broil-ers were fed the high-protein diet. Protease im-proved CP and fat digestibilities (Table 6). The determined AME was higher when the high-pro-tein and high-energy diets were fed, but protease had no effect on AME. An interaction occurred between protein and energy in which CP, fat, and energy digestibilities as well as determined AME improved as energy increased in the low-protein diets, with the reverse effect occurring in the high-protein diets. An interaction between

protein and protease was observed in which digestibilities of CP and energy and the deter-mined AME were greater when the protease was added to the high-protein diets as compared with the low-protein diets. An interaction between energy and protease was also associated with a greater increase in energy digestibility and AME when protease was added to the high-energy di-ets, as compared with the low-energy diets.

DISCUSSION

Currently, the enzyme most commonly used in broiler feeds is phytase. Using a second or

Table 6. Ileal digestibility values of CP, fat, and energy and determined AME values of male broilers at 42 d of age fed 2 levels of protein and energy, with or without protease, experiment 21

Item

Digestibility, %AME, kcal/kgCP Fat Energy

Treatment2

Low protein, low energy, no protease 78.9bc 76.5d 76.8abc 3,465bc

High protein, low energy, no protease 81.9a 80.5bcd 79.3a 3,577ab

Low protein, high energy, no protease 82.2a 83.6abc 78.0ab 3,523ab

High protein, high energy, no protease 78.4c 80.6bcd 74.4c 3,441bc

Low protein, low energy, protease 78.9bc 79.5cd 75.2bc 3,345c

High protein, low energy, protease 82.0a 84.9ab 79.1a 3,512ab

Low protein, high energy, protease 83.8a 86.1a 77.4abc 3,553ab

High protein, high energy, protease 81.3ab 85.3a 78.0ab 3,635a

SEM 0.62 1.07 0.67 31.74

Main effect Protein Low 80.4 80.4 76.9 3,471 High 81.4 83.9 77.7 3,541 Energy Low 80.9 81.4 77.6 3,475 High 80.9 82.9 77.0 3,538 Protease No 80.3 80.3 77.1 3,501 Yes 81.5 84.0 77.4 3,511

P-valueSource of variation Protein 0.0240 0.0001 0.1005 0.0029 Energy 0.9284 0.0688 0.2144 0.0067 Protease 0.0101 0.0001 0.5476 0.6705 Protein × energy 0.0001 0.0001 0.0001 0.0030 Protein × protease 0.0159 0.9850 0.0050 0.0176 Energy × protease 0.3878 0.2339 0.0157 0.0001 Protein × energy × protease 0.4320 0.7855 0.1541 0.2356a–dMeans within a column not sharing a common superscript differ (P < 0.05).1Means were based on 5 birds taken randomly from 8 randomly selected replicate pens per treatment.2Treatments: high and low CP and ME (differences of 7 and 3%, respectively), with or without protease at 200 ppm (15,000 protease units/kg).


third mono-component enzyme, as well as a mixed enzyme product besides phytase, is of in-terest to broiler integrators because of the possi-bility of improving digestibilities and the result-ing potential savings in feed formulation. Amino acids are costly nutrients that (with the exception of their synthetic forms) originate from dietary proteins. Protein degradation is largely mediated by gastric and pancreatic secretions [29]. Pro-tein digestibility is variable between ingredients used in broiler feeds [1, 8]; therefore, undigested or incompletely digested protein represents an important opportunity for use of an exogenous protease in broiler feeds.

In both studies presented here, the supple-mentation of a mono-component protease had no effect on BWG. However, a quadratic beneficial FE response was observed as graded levels of protease were added in the first study. In the sec-ond study, an improvement in FE was also ob-served when the protease was added to the low-protein or high-protein diets at the commercially recommended dose of 200 ppm. Research on the use of mono-component proteases is scarce, and reported responses have been very enzyme spe-cific. Ghazi et al. [14, 15] reported on the use of different mono-component proteases. In one study [14], soybean meal was pretreated with 2 mono-component proteases and fed to broilers. One of the enzymes resulted in no change in per-formance, whereas the other protease had a posi-tive effect on both BWG and FE. In a separate study, Ghazi [15] reported that the use of one mono-component protease resulted in increased BWG and FI, but that FE was either negatively affected or not affected at all, depending on the protease concentration used. Persia et al. [30], working with turkeys and using a multienzyme complex containing protease, reported no differ-ences in BWG but improvements in FE from 9 to 15 wk of age.

As with any other protease, AA derived from its protein degradation activity are expected to become available for intestinal absorption, de-pending, in part, on the affinity between the enzyme and substrate. For instance, trypsin, a serine protease released from the pancreas, will preferentially cleave AA bonds that have lysine and arginine. The mono-component protease used in the present studies was also a serine protease; however, its preferable peptide bond

cleavage site has not yet been defined. The re-ductions in CP and AA between the positive control and negative control diets in experi-ment 1, as well as between the high-protein and low-protein diets in experiment 2, were chosen to provide enough room for any benefit arising when the protease was added.

The improvement in FE observed with the addition of the protease appeared to be mediated through an improved AA availability balance because the protease had no effects on BWG or FI. This better available AA balance would have improved body muscle deposition and thus the conversion of feed to gain. This was further supported by an increased apparent digestibility of CP when the protease was added to the high-protein diets. In addition, the apparent digest-ibilities of fat and energy and the determined AME were also improved when the protease was added to the high-protein and high-energy diets in experiment. 2. A better digestible AA balance would be expected to result in carcass composition changes, such as greater breast yields or lower abdominal fat pad weights, but no changes were observed in experiment 1, and carcass composition was not measured in ex-periment 2. The authors could find no published work reporting the effect of mono-component proteases on carcass yields.

In the current research, an improvement of 1.8% in CP digestibility was observed when the protease was added to the high-protein di-ets, whereas an improvement of only 1% was seen in the low-protein diets. Cowieson and Ravindran [31] reported that the inclusion of a multienzyme complex containing xylanase, pro-tease, and amylase improved N digestibility by 2.5%, which when converted to CP digestibility, represented an improvement of 15.6%. This im-provement was accompanied by a 5.5% greater BWG and a 4% improvement in FE in broilers fed corn-soybean meal diets. They formulated the low-nutrient diets to contain 5.2% less ME, with the same protein but lower in lysine (7% re-duction) and TSAA (15% reduction) and higher in arginine (3% increase) and threonine (4.5% increase). This differs from the current studies, in which no changes in ME were formulated and protein, lysine, and TSAA were decreased. Gra-cia et al. [32] reported no effect on any broiler performance measure from 1 to 21 d of age and


a 4.2% improvement in N retention when a mul-tienzyme complex containing the same enzymes as those used by Cowieson and Ravindran [31] was used in corn-, soybean meal-, and wheat middlings-based diets. The variability in results from published research with mono-component proteases and multienzyme complexes is evi-dent. This variability is present even when a similar multienzyme complex is used [31, 32].

CONCLUSIONS AND APPLICATIONS

1. The commercial mono-component pro-tease tested in these studies had a benefi-cial effect on FE but no effect on other performance measures.

2. The mono-component enzyme improved CP and fat digestibilities, and this effect was more pronounced in the high-pro-tein diets.

3. No effects of the protease on carcass yields were observed.

REFERENCES AND NOTES

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2. Wang, X., and C. M. Parsons. 1998. Effect of raw ma-terial source, processing systems, and processing tempera-tures on amino acid digestibility of meat and bone meals. Poult. Sci. 77:834–841.

3. Araba, M., and N. M. Dale. 1990. Evaluation of pro-tein solubility as an indicator of overprocessing soybean meal. Poult. Sci. 69:1749–1752.

4. Parsons, C. M., K. Hashimoto, K. J. Wedeking, Y. Han, and D. H. Baker. 1992. Effect of overprocessing on availability of amino acids and energy in soybean meal. Poult. Sci. 71:133–140.

5. Douglas, M. W., C. M. Parsons, and M. R. Bedford. 2000. Effect of various soybean meal sources and Avizyme on chick growth performance and ileal digestible energy. J. Appl. Poult. Res. 9:74–80.

6. Goldflus, F., M. Ceccantini, and W. Santos. 2006. Amino acid content of soybean samples collected in dif-ferent Brazilian states—Harvest 2003/2004. Braz. J. Poult. Sci. 8:105–111.

7. de Coca-Sinova, A., D. G. Valencia, E. Jiménez-Moreno, R. Lázaro, and G. G. Mateos. 2008. Apparent ileal digestibility of energy, nitrogen, and amino acids of soybean meals of different origin in broilers. Poult. Sci. 87:2613–2623.

8. Lemme, A., V. Ravindran, and W. L. Bryden. 2004. Ileal digestibility of amino acids in feed ingredients for broilers. World’s Poult. Sci. 60:423–437.

9. Nir, I., Z. Nitsan, and M. Mahagna. 1993. Compara-tive growth and development of the digestive organs and of

some enzymes in broiler and egg type chicks after hatching. Br. Poult. Sci. 34:523–532.

10. Le Huerou-Luron, I., E. Lhoste, C. Wicker-Planquart, N. Dakka, R. Toullec, T. Corring, P. Guilloteau, and A. Puig-server. 1993. Molecular aspects of enzyme synthesis in the exocrine pancreas with emphasis on development and nutri-tional regulation. Proc. Nutr. Soc. 52:301–313.

11. Marsman, G. J., H. Gruppen, A. F. van der Poel, R. P. Kwakkel, M. W. Verstegen, and A. G. Voragen. 1997. The effect of thermal processing and enzyme treatments of soy-bean meal on growth performance, ileal nutrient digestibili-ties, and chyme characteristics in broiler chicks. Poult. Sci. 76:864–872.

12. Naveed, A., T. Acamovic, and M. R. Bedford. 1998. Effect of enzyme supplementation of UK-known Lupinis al-bus on growth performance in broiler chickens. Br. Poult. Sci. 39:S36–S37.

13. Simbaya, J., B. A. Slomminski, W. Guenter, A. Mor-gan, and L. D. Campbell. 1996. The effects of protease and carbohydrase supplementation on the nutritive value of canola meal for poultry. Anim. Feed Sci. Technol. 61:219–234.

14. Ghazi, S., J. A. Rooke, H. Galbraith, and M. R. Bed-ford. 2002. The potential for the improvement of the nutri-tive value of soya-bean meal by different proteases in broiler chicks and broiler cockerels. Br. Poult. Sci. 43:70–77.

15. Ghazi, S., J. A. Rooke, and H. Galbraith. 2003. Im-provement of the nutritive value of soybean meal by prote-ase and α-galactosidase treatment in broiler cockerels and broiler chicks. Br. Poult. Sci. 44:410–418.

16. Mahagna, M., I. Nir, M. Larbier, and Z. Nitsan. 1995. Effect of age and exogenous amylase and protease on devel-opment of the digestive tract, pancreatic enzymes activities and digestibility of nutrients in young meat-type chickens. Reprod. Nutr. Dev. 35:201–212.

17. Ronozyme ProAct CT, obtained from Novozymes A/S, Bagavaerd, Denmark.

18. White, J. A., R. J. Hart, and J. C. Fry. 1986. An evalu-ation of the waters pico-tag system for the amino-acid-anal-ysis of food materials. J. Automat. Chem. 8:170–177.

19. Hagen, S. R., B. Frost, and J. Augustin. 1989. Precol-umn phenylisothiocyanate derivatization and liquid-chro-matography of amino-acids in food. J. Assoc. Off. Anal. Chem. 72:912–916.

20. Doux-Frangosul, Montenegro, Rio Grande do Sul, Brazil.

21. Rostagno, H. S., L. F. T. Albino, J. L. Donzele, P. C. Gomes, R. F. Oliveira, D. C. Lopes, A. S. Ferreira, and S. L. T. Barreto. 2005. Tabelas Brasileiras para Aves e Suínos. Composição de Alimentos e Exigências Nutricionais. 2nd ed. UFV, Viçosa, Minas Gerais, Brazil.

22. AOAC. 2000. Official Methods of Analysis. 17th ed. Assoc. Off. Anal. Chem., Arlington, VA.

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