Mitigating the Effects of Halofuginone on Skin Strength by Feeding L-Proline to Broiler Chickens1,2

METABOLISM AND NUTRITION Mitigating the Effects of Halofuginone on Skin Strength by Feeding L-Proline to Broiler Chickens1'2 K. D. CHRISTENSEN,3 N. G. ZIMMERMANN,4 C. L. WYATT, and T. N. GOODMAN Washington State University, Puyallup Research and Extension Center, 7612 Pioneer Way E., Puyallup, Washington 98371-4998 R. J. BUHR Department of Poultry Science, University of Georgia, Athens, Georgia 30602 P. F. TWINING Recon Inc., Taylors Island, Maryland 21669 ABSTRACT Two experiments were conducted to determine whether supplemental levels of L-proline in the diets of broiler chickens would mitigate the skin weakening effect caused by continuous feeding of the anticoccidial halofuginone. In Experiment 1, tensile strength and collagen levels in thigh apteria skin were determined at 21 and 42 d of age in male broilers fed 0, .5, and 1% L-proline with either halofuginone (3 mg/kg) or salinomycin (61 mg/kg). In Experiment 2, the same measurements were made on female broilers receiving diets containing halofuginone and supplemented with 0, .5, or 1% L-proline, 1% L-proline through 21 d of age, or 1% L-glutamic acid through 21 d of age, or a diet containing high L-proline feedstuffs (corn gluten meal and ring dried blood meal). In Experiment 1, dermis thickness of thigh apteria was measured in the males at Day 21. Skin strength was increased in male and female broilers fed halofuginone with addition of .5 and 1% L-proline, respectively, at 21 and 42 d of age. Continuous incorporation of synthetic L-proline into diets was shown to improve skin strength in females, whereas diets formulated to contain high levels of L-proline from feedstuffs, 21-d feeding of L-proline, or L-glutamic acid did not increase skin strength. (Key words: skin strength, L-proline, glutamic acid, halofuginone, collagen) 1995 Poultry Science 74:1610-1621

INTRODUCTION Received for publication April 13, 1994. Accepted for publication May 2, 1995. X A portion of these data was presented at the 79th Annual Meeting of the Poultry Science Association, Inc., August, 1991, College Station, TX. 2 Project Number 0979. Animal Sciences Scientific Paper Number 8083. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164. 3 Current address: Draper Valley Farms, P.O. Box 838, 1000 Jason Lane, Mount Vernon, WA 98273. 4 To whom correspondence should be addressed. Current address: Lower Eastern Shore Research and Education Center, University of Maryland, 11990 Strickland Dr., Princess Anne, MD 21853.

Continuous feeding of the anticoccidial drug halofuginone to broiler chickens has been associated with lower skin tensile strength, increased incidence of skin tearing during processing, a thinner dermal layer of the skin, and lowered skin collagen levels (Angel et al, 1985; Casey et al, 1989,1992; Granot et al, 1991a; Crosley et al, 1992, Christensen et al, 1994; and Zimmermann et al, 1994). In vitro studies have demonstrated that halofuginone in-

1610

PROLINE AND SKIN STRENGTH IN BROILERS

terferes with production of collagen (Granot et al, 1991b). Collagen protein is unique in its synthesis, composition, and structure. The collagen molecule consists of three a chains in a triple helix configuration. Collagen contains 35% glycine, 12% L-proline, and 10% hydroxyproline. Hydroxyproline is not incorporated into the collagen molecule, but specific L-proline moieties are hydroxylated after being incorporated into procollagen chains (Kuhn, 1987). The hydroxylation of incorporated L-proline requires ascorbic acid as a reducing agent (Blank and Peterkofsky, 1975). The reaction also requires other cofactors, such as iron (Kao et ah, 1975), copper (Stryer, 1975), and oxygen (Uitto and Prockop, 1974). Identifying possible disruptions of L-proline incorporation or hydroxylation could reveal the mode of action of halofuginone on collagen synthesis. Smith et al. (1977) reported an increase in total collagen concentration when supplemental ascorbic acid was fed to broiler chickens. However, Zimmermann et al. (1994) did not observe any mitigating effect of added ascorbic acid on skin tensile strength of broilers fed halofuginone. Proline analogs interfere with the incorporation of L-proline into procollagen during chain assembly, decrease the thermal stability of collagen, and prevent formation of its triple helical conformation (Uitto and Prockop, 1974). If halofuginone is acting as a L-proline analog, then increased dietary L-proline may reduce its skin-weakening effect by competitive exclusion. L-proline is considered to be a nonessential amino acid for chickens and is rarely considered in the formulation of practical diets. Two experiments were conducted to determine whether increased dietary Lproline or its precursor L-glutamic acid

1611

(Shen et al., 1973) mitigate the skinweakening effect of continuous administration of halofuginone. MATERIALS AND METHODS Housing and Management In each experiment, 720 Peterson®5 x Hubbard®6 chicks were raised in a lightand temperature-controlled brooder house. Twenty chicks were brooded and reared in 36 identical 1.23 x 3.68 m pens. Biaxial fluorescent tubes provided 10 lx illumination for 23 h/d. Feed and water were available for ad libitum consumption. The chicks were started under radiant tube brooders on fir shavings with a feeder flat plus adjustable water lines having six nipple drinkers per pen. At 7 d of age, a hanging tube feeder (41 cm diameter) was installed and the feeder flats removed. Male chicks were used in Experiment 1 and female chicks were used in Experiment 2. Diets In Experiment 1, six factorially arranged dietary treatments were prepared by adding anticoccidial (halofuginone, 3 mg/kg or salinomycin, 61 mg/kg) and L-proline7 (0, .5, or 1.0%) to basal starter and grower diets (Table 1). In Experiment 2, five dietary treatments were prepared by adding halofuginone (3 mg/kg) and L-proline (0, .5, or 1.0%) or 1.0% L-glutamic acid to either starter or grower basal diets. In addition, feed formulated with corn gluten meal and ring dried blood meal to yield an endogenously elevated L-proline diet was fed with halofuginone (Table 1). Starter and grower diets were fed from Day 1 to 21 and Day 22 to 42, respectively. The grower diet, fed from Day 38 to 42, excluded the anticoccidial drugs. The diets were commercially formulated, with proprietary restrictions, using current industry standards for essential amino acid requirements and they were fed as a mash.

5

Peterson Industries, Decatur, AR 72722. Hubbard Farms, Walpole, NJ 03608. United States Biochemical Corp., Cleveland, OH 44101. 8 Woodson-Tenent Laboratories, Inc., in Des Moines, IA and Memphis, TN 38101 for Experiments 1 and 2, respectively. 6

Measurements All diets were analyzed for protein, Lproline, and glutamic acid levels8 using procedures described by AOAC (1990)

1612

CHRISTENSEN ET AL. TABLE 1. Ingredients and analyses of diets fed, Experiments 1 and 21 Experiment 2 High-L-prolinii feedstuffs

Basal diets Ingredients and analyses

Starter

Grower

Starter

Grower

*""„> Anticoccidial2 Corn Soybean meal, 48.5% Corn gluten meal, 60% Wheat, soft white Meat and bone meal Blood meal, ring dried Blended fat DL-methionine, 99% L-lysine HC1, 98% Tri-calcium phosphate, 18/32 Limestone Salt Copper sulfate Choline, Chl-60 Trace ingredients3 Calculated ME, kcal/kg Protein, % TSAA, % Lysine, % Proline, % Analyzed, Experiment 1 Protein, % Proline, % Analyzed, Experiment 2 Protein, % Proline, %

.05 35.05 27.40

.05 45.63 22.82

25.00 5.00

20.00 5.00

5.00 .165 .035 1.10 .45 .25 .100 .055 .390

4.23 .15 .015 1.10 .38 .26 .50 .050 .275

3,146 21.45 .92 1.19 1.18

3,190 19.91 .84 1.06 1.24

21.85 1.22

20.26 1.15

21.23 1.07

19.86 1.18

.05 51.29 22.99 5.00 10.00 3.00 .50 3.23 .07 1.25 .32 .25 .10 .025 .411

.05 40.60 19.77 5.00 25.00 3.00 1.00 4.67 .08 .017 1.30 .33 .25 .05 .067 .438

3,146 22.52 .89 1.11 1.56

3,256 20.76 .80 1.00 1.49

22.63 1.42

20.12 1.28

Starter and grower diets were fed from Day 1 to 21 and Day 22 to 42, respectively. Grower diet without anticoccidial was fed from Day 38 to 42. 2 Dependent on treatment, either halofuginone (3 mg/kg) or salinomycin (61 mg/kg) was used as the anticoccidial drug. 3 Levels of trace ingredients including vitamins, minerals, and growth promoters are proprietary.

method 928.30. Group BW and feed consumption were determined in each pen on Day 21 and 42. Mean BW and feed conversion ratio (grams of feed consumed:grams of BW gain) were calculated for each pen. On Day 21 and 42, five birds per pen were selected at random, killed by cervical dislocation, and tensile strength measurements made on fresh, defeathered 1-cm sections of thigh apteria skin (Christensen et ah, 1994). All samples were collected from

the same site on the left thigh. Pen means were used for statistical analysis. On Day 43, the remaining birds in each treatment were grouped, commercially processed, and the percentage of skin tears was calculated (Christensen et ah, 1994). The same individual recorded skin tear data in both experiments and was aware of the treatments. Only the back and thigh regions were examined for tears. In Experiment 1, skin samples taken from the thigh aptera on Day 21 were fixed in 10% formalin.9 Transverse skin sections, 5 mm thick, were dehydrated through a graded series of ethanol9 (70 to9 100%), 9 Fisher Scientific Co., Pittsburgh, PA 15129. 10 Data Sheet No. 123B, Instructions for the JB-4 cleared in two changes of xylene (100%), in 95% ethanol, and embedded Embedding Kit, Polysdences, Inc., Warrington, PA rehydrated in JB-410 plastic. Sections were cut at 8 (im, 18976.

1613

PROLINE AND SKIN STRENGTH IN BROILERS

TABLE 2. Measured L-proline and L-glutamic acid in dietary treatments fed, Experiments 1 and 21 Experiment number

L •proline

L-glutamic acid

Anticoccidial

L-proline added

Starter

Grower

Halofuginone Salinomycin Halofuginone Salinomycin Halofuginone Salinomycin Halofuginone Halofuginone Halofuginone Halofuginone Halofuginone Halofuginone

0 0 .5 .5 1.0 1.0 0 .5 1.0 1.02 03 04

1.18 1.26 1.81 1.62 2.27 2.34 1.07 1.40 1.63 1.75 1.42 1.14

1.19 1.10 1.60 1.58 1.89 2.12 1.18 1.72 1.97 1.18 1.28 1.25

Starter

Grower

3.81 3.62 3.80 3.74 3.55 3.76 3.80 3.83 3.72 4.43 4.04 5.04

3.44 3.54 3.67 3.52 3.52 3.83 4.18 3.94 4.17 4.18 3.77 4.20

(%) 1

2

1

Analysis of diets for Experiment 1 were done by Woodsen-Tenent Laboratories, Inc., Des Moines, IA 50305. Analysis of diets for Experiment 2 were done by Woodsen-Tenent Laboratories, Inc., Memphis, TN 38101. 2 Basal diets were fed with 1.0% L-proline added to the starter diet only (1 to 21 d of age). 3 Diets formulated to contain higher levels of L-proline from feedstuffs. 4 Basal diets were fed with 1.0% L-glutamic acid added to the starter diet only (1 to 21 d of age).

floated onto glass slides, and dried on slide warmer at 50 C. Slides were stained and dermis thickness measured as described by Christensen et al. (1994). In both experiments, skin samples collected on Day 21 and 42 were frozen (-20 C) and subsequently analyzed for collagen using spectrophotometry analysis of hydroxyproline (Christensen et al., 1994).

1987). Tukey's multiple comparison procedure (Steel and Torrie, 1980) was used to compare treatments using P < .05 as the level of significance. RESULTS

Calculated and analyzed nutrients in the basal diets fed in both experiments are shown in Table 1. Table 2 contains the analyzed dietary levels for L-proline and Statistical Analysis L-glutamic acid in each diet fed. CompariIn Experiment 1, BW, feed conversion, son of calculated and analyzed values skin tensile strength, skin dermis thickness, shows that L-proline added to the basal and skin collagen content were analyzed as diets in both experiments was reflected in a 2 x 3 factorial ANOVA with anticoccidial the mixed rations. drug and level of added L-proline as the main effects. These data were also analyzed Growth Performance by one-way ANOVA to separate treatment means if significant main effect interaction In Experiment 1, the addition of .5 or 1% (P < .05) occurred. Pearson's correlation L-proline to the diet of male broilers caused coefficient between dermis thickness and a significant increase in BW at 21 d of age skin tensile strength was calculated. In (Table 3). There was also an improvement Experiment 2, one-way ANOVA was used in feed conversion with the addition of Lto detect treatment differences on all meas- proline (P < .0001). Anticoccidial had no ured variables. Pearson's correlation coeffi- effect on feed conversion at 21 d of age but cient between percentage skin tears and halofuginone increased BW (P = .047) skin tensile strength was calculated. All compared to salinomycin. Treatment differdata analysis utilized the General Linear ences in BW and feed conversion were not Models procedure of SAS® (SAS Institute, significant at 42 d of age. However, there

1614

CHRISTENSEN ET AL.

was a trend toward higher BW (P = .072) Day 21 Day 42 when L-proline was incorporated and more efficient feed utilization (P = .085) when salinomycin was incorporated into the diets. No main effect interactions for any growth performance variable occurred at 21 or 42 d of age (Table 3). In Experiment 2, BW of 21-d-old female broilers was depressed (P < .05) when fed the L-glutamic acid or high-L-proline feedstuffs diets compared with pullets fed diets supplemented with L-proline (Table 4). 0 .5 1 0 .5 1 Compared with the control diet, feed PERCENT ADDED PROLINE conversion at 21 d of age was not affected by addition of L-proline to the feed but the FIGURE 1. Interaction between anticoccidial and high-L-proline feedstuffs had inferior feed added L-proline on skin tensile strength at 21 and 42 d conversion. The dietary treatments did not of age, Experiment 1. Each salinomycin (•) and affect pullet BW (P = .260) or feed conver- halofuginone (•) data point represents the 5c ± SEM of sion (P = .082) at 42 d of age. the skin tensile strength from six replicate pens, each providing an average strength from five randomly No coccidial lesions were observed dur- pen sampled birds. Data points, within age, not sharing a ing random intestinal examinations in ei- common letter differ significantly (P £ .05). ther experiment. Skin Tensile Strength In Experiment 1, addition of .5 and 1% Lproline to the male broiler diet increased skin strength 10 and 12% at 21 d of age compared with the diet containing no added L-proline (Table 5). Skin strength at Day 42 was increased by 13% in males fed 1% added L-proline compared with the basal diet. Skin tensile strength data in Experiment 1 had significant anticoccidial by added L-proline interactions at 21 and 42 d of age (Table 5 and Figure 1). No level of supplementary dietary L-proline had an effect on skin strength of male broilers fed salinomycin at either 21 or 42 d of age. However, the increase in skin strength in males fed halofuginone was dependent upon level of added L-proline at 21 and 42 d of age. In Experiment 2, L-proline added at .5 and 1% levels increased skin strength in females fed halofuginone by 29 and 39% at Day 21 (Table 6). Pullets fed these diets also had higher skin strength than the highproline feedstuffs diet and the L-glutamic diet at 21 d of age. Unexplainably, adding 1% L-proline only in the starter diet did not increase skin strength compared to the basal diet at 21 d of age. Addition of 1% Lproline in both the starter and finisher diets increased skin strength 29% compared with

the no added proline diet at Day 42. Skin strength of females fed the remaining diets was intermediate. Dermis Thickness Dermis thickness was increased in 21-d-old male broilers by addition of .5% Lproline to the diet but 1% added L-proline did not cause further dermal thickening over the .5% L-proline addition (Table 5). Dermis thickness was suppressed by the continuous feeding of halofuginone (P = .0001). The interaction of main effects was not significant. Dermis thickness was correlated to skin strength in 21-d-old male broilers (r = .9509; P = .0036; n = 6). Skin Tearing During Processing In Experiment 1, the percentage of skin tears observed on male broilers at 43 d of age after scalding and feather removal was 8.9 on birds fed the continuous halofuginone diet without added L-proline (Table 5). Skin tears were not seen on birds in any of the other treatments in Experiment 1. In Experiment 2, the percentage of skin tears observed was 25.0,11.7, and 7.0 in the 0, .5, and 1.0% added L-proline treatments, respectively (Table 6). Adding 1.0% L-

PROLINE AND SKIN STRENGTH IN BROILERS

1615

TABLE 3. Effect of added dietary L-proline and anticoccidial on male broiler performance at 21 and 42 d of age, Experiment l 1 Day 21 Anticoccidial

Proline added

BW

0 0 .5 .5 1.0 1.0 Pooled SEM P =

829
&g) 1.403* 1.416» 1.359b 1.355b 1.350b 1.362b .010 .0001

(g) 2,345 2,323 2,401 2,393 2,385 2,426 32

824b 861» 865" 6 .0001 857» 842b 5 .0469

1.409' 1.357b 1.355b .007 .0001 1.370 1.380 .006 .2702

2,334 2,397 2,403 23

.8729

.6733

Feed conversion2

(s) Halofuginone Salinomycin Halofuginone Salinomycin Halofuginone Salinomycin

Main effects3

0

Added proline, %

in

Anticoccidial

Day 42

1.0 Pooled SEM P = Halofuginone Salinomycin Pooled SEM P =

Interaction of main effects

P =

Feed conversion 2

BW

.2789

(g=g) 1.762 1.748 1.755 1.730 1.747 1.724 .014 .4285

.8976

1.754 1.744 1.736 .010 .3867 1.754 1.735 .008 .0848

.6053

.9139

.0724 2,377 2,377 18

a_c

Means within a column and treatment main effect with no common superscript differ significantly (P £ .05). *Each of the L-proline and anticoccidial main effect means represent 12 and 18 replicate pens, respectively. 2 Feed conversion ratios (grams feed:grams BW gain) are cumulative from Day 1. 3 L-proline and the anticoccidials (halofuginone, 3 mg/kg and salinomycin, 61 mg/kg) were added to basal diets in a factorial arrangement.

TABLE 4. Effect of elevated L-proline levels and halofuginone on female broiler performance at 21 and 42 d of age, Experiment 2 Day 21 Treatment

BW

(g) 0% added L-proline .5% added L-proline 1.0% added L-proline 1.0% added L-proline2 High L-proline feedstuffs3 1.0% added L-glutamic acid4 Pooled SEM P =

653ab 677» 667» 682" 616b 634b 7 .0001

Feed conversion 1 (g:g) 1.530b 1.512b 1.524b 1.517b 1.625a 1.581* .020 .0024

Day 42 BW

Feed conversion 1

(g)

(g=g)

1,818 1,861 1,884 1,843 1,850 1,826 20

1.851 1.873 1.854 1.948 1.840 1.873 .024 .0819

.2596

"-bMeans within a column with no common superscript differ significantly (P £ .05). x Feed conversion ratios (grams feed:grams BW gain) are cumulative from Day 1. 2 Basal diets were fed with 1.0% L-proline added to the starter diet only (1 to 21 d of age). 3 Diet formulated to contain high level of L-proline from feedstuffs. 4 Basal diets were fed with 1.0% L-glutamic acid added to the starter diet only (1 to 21 d of age).

1616

CHRISTENSEN ET AL.

TABLE 5. Skin tensile strength and dermis thickness of 21-d-old male broilers, skin tensile strength of 42-d-old male broilers, and skin tears observed during processing when fed different L-proline and anticoccidial diets, Experiment l 1 Day 21 Anticoccidial Halofuginone Salinomycin Halofuginone Salinomycin Halofuginone Salinomycin Main effects2 Added L-proline, %

Anticoccidial

Interaction of main effects

Added L-proline

Skin strength

Dermis thickness

Day 42 Skin strength

Day 43 Skin tears

(%) 0 0 .5 .5 1.0 1.0 Pooled SEM P =

(g/cm) 2,744' 3,488»b 3,1351* 3,848" 3,394* 3,524* 132 .0001

(mm) .1241' .1327bc .1306* .1444' .1325* .1352* .0020 .0001

(g/cm) 3,643b 4,667* 4,103* 4,861* 4,648" 4,693" 181 .0005

(%) 8.9 0 0 0 0 0

0 .5 1.0 Pooled SEM P = Halofuginone Salinomycin Pooled SEM P =

3,116b 3,491" 3,459" 93 .0139 3,091b 3,620" 76 .0001

.1284b .1375" .1339" .0014 .0004 .1291b .1374' .0017 .0001

4,155b 4,447,* 4,668" 128 .0302 4,131b 4,735" 107 .0004

P =

.0458

.0388

.2056

"-•Means within a column and treatment main effect with no common superscript differ significantly (P <, .05). !Each of the L-proline and anticoccidial main effect means represents 12 and 18 replicate pens, respectively, with 5 birds per pen and 21 dermis thickness measurements per bird. Pen means were used for statistical analysis. Birds remaining in replicate pens were pooled within treatment and commercially slaughtered to calculate percentage skin tears (n = 50 to 55). 2 L-proline and the anticoccidials (halofuginone, 3 mg/kg and salinomycin, 61 mg/kg) were added to basal diets in a factorial arrangement.

TABLE 6. Skin tensile strength of 21- and 42-d-old female broilers and percentage skin tears observed during processing when fed various levels of L-proline or L-glutamic acid, Experiment 21 Treatment

Day 21 Skin strength

0% added L-proline .5% added L-proline 1.0% added L-proline 1.0 % added L-proline2 High-proline feedstuffs3 1.0% added L-glutamic acid 4 Pooled SEM P =

1,552b 1,997" 2,156" 1,797* 1,542b 1,549b 93 .0001

Day 42 Skin strength (g/cm) 2,550b 2,812* 3,286" 2,684"b 2,647* 2,585* 164 .0371

Day 43 Skin tears (%) 25.0 11.7 7.0 11.6 13.7 14.5

"-bMeans within a column with no common superscript differ significantly (P <, .05). 'Each of the treatment means represent six replicate pens with five birds per pen. Pen means were used for statistical analysis. Birds remaining in replicate pens were pooled within treatment and commercially slaughtered to calculate percentage skin tears (n = 50 to 55). 2 Basal diets were fed with 1.0% L-proline added to the starter diet only (1 to 21 d of age). 3 Diet formulated to contain high level of L-proline from feedstuffs. 4 Basal diets were fed with 1.0% L-glutamic acid added to the starter diet only (1 to 21 d of age).

P =

P =

0 .5 1.0 Pooled SEM P = Halofuginone Salinomycin Pooled SEM

0 0 .5 .5 1.0 1.0 Pooled SEM P =

(%)

Added proline

.8483

134* 10* 131* 9 .0183 110b 133* 7 .0264

126* 142* 88b 112* 116* 146* 12 .0263

Soluble

.9452

.2497 134b 156* 5 .0033

137 147 151 6

126b 149* 137* 156* 139* 164* 9 .0430

Insoluble

Collagen content Day 21

.7882

271* 247b 282* 8 .0157 244b 298* 7 .0001

251bc 290* 225c 269*c 255bc 309* 12 .0003

Total

.3

150 148 157 7 .5 147 157 5 .2

• (mg/g) 135 162 149 148 155 160 9 .4

Solu

'"Means within a column and treatment main effect with no common superscript differ significantly (P 1 Collagen content of dried, defatted skin samples. 2 L-proline and the anticoccidials (halofuginone, 3 mg/kg and salinomycin, 61 mg/kg) were added to b

Interaction of main effects

Anticoccidial

Main effects2 Added proline, %

Halofuginone Salinomycin Halofuginone Salinomycin Halofuginone Salinomycin

Anticoccidial

TABLE 7. Effect of diet on skin collagen content1 in of 21- and 42-d-old male

1618

CHRISTENSEN ET AL. TABLE 8. Effect of diet on skin collagen content1 in of 21- and 42-d-old female broilers when fed different dietary treatments, Experiment 2 Collagen content Day 21

Collagen content Day 42

Treatment

Soluble

Insoluble

Total

Soluble

Insoluble

Total

0% added L-proline .5% added L-proline 1.0% added L-proline 1.0% added L-proline2 High L-proline feedstuffs3 1.0% added L-glutamic acid4 Pooled SEM

151 121 158 182 146 156 14

129 121 135 160 115 114 11

280"'' 241b 292ab 342" 260* 271*

99 100 107 100 106 109 8

147 129 148 144 143 138 7

246 229 256 244 249 247 11

.1325

.1060

19 .0263

.9134

.3132

.6611

ab

' Means within a column and treatment main effect with no common superscript differ significantly (P £.05). Collagen content of dried, defatted skin samples. 2 Basal diets were fed with 1.0% L-proline added to the starter diet only (1 to 21 d of age). 3 Diet formulated to contain high level of L-proline from feedstuffs. 4 Basal diets were fed with 1.0% L-glutamic acid added to the starter diet only (1 to 21 d of age). 1

proline or L-glutamic acid to the diets for et al, 1962). At 21 d of age BW and feed the first 21 d resulted in 11.6 and 14.5% skin conversion were improved with the additears. The high-L-proline feedstuffs treat- tion of L-proline to the commercially ment yielded 13.7% skin tears. The correla- formulated basal diets fed in these experition coefficient (r) between percentage of ments. Because BW and feed conversion skin tears and skin strength was -.7277 (P = ratios were similar to previous flocks grown in this facility we have no reason to .1011; n = 6). suspect that any essential nutrient was limited in the basal diets. Perhaps genetic Skin Collagen changes in modern commercial broilers In Experiment 1, addition of L-proline to have increased the need for L-proline in the diets of males significantly affected the diet. soluble and total skin collagen content at Roy and Bird (1959) reported that 1% Day 21, but not in a dose-dependent additional L-proline improved growth manner (Table 7). No level of L-proline had performance in chicks treated with /3an effect on skin collagen at 42 d of age. aminopropionitrile fumarate (BAPN). Halofuginone reduced insoluble and total Birds fed BAPN are prone to ruptured skin collagen at 21 and 42 d of age. Soluble aorta, pulmonary hemorrhage, and leg collagen was also reduced at 21 d of age. In deformities. These symptoms are related Experiment 2, the 1% added L-proline diet to collagen formation. /3-Amino-propiofed for 21 d significantly increased total nitrile fumarate is a potent irreversible collagen at Day 21 compared to continuous inhibitor of lysl oxidase (Wilmarth and inclusion of .5% L-proline, other treatments Froines, 1992). Lysl oxidase catalyzes lywere intermediate (Table 8). No other sine in elastin and collagen to initiate cross-linking in these proteins (Reiser et differences were observed. al, 1992). Although supplemental Lproline did not mitigate the BAPNDISCUSSION induced symptoms (Roy and Bird, 1959), Although L-proline is not considered to perhaps both BAPN and halofuginone be an essential amino acid in practical affect chickens by similar mechanisms and broiler diets, chicks fed purified amino the dietary requirement for L-proline is acid diets require .5% L-proline for op- increased when collagen formation is distimal growth and feed efficiency (Greene turbed.

PROLINE AND SKIN STRENGTH IN BROILERS

The results have demonstrated that skin tensile strength in broiler chickens continuously fed halofuginone can be increased by supplementing the diet with Lproline. The effective level of L-proline supplementation varied between the sexes. Skin strength was increased and skin tearing in males was eliminated by addition of .5% L-proline, whereas females had increased skin strength and exhibited 7% skin tears when 1% supplemental Lproline was fed. The dose-dependent improvement of skin tensile strength and percentage of skin tearing between 0, .5, and 1% added L-proline in females fed halofuginone suggests that further improvement in skin strength may be achieved with supplementation of Lproline above 1%. Christensen et al. (1994) reported a highly significant inverse relationship between skin strength and percentage skin tears. Although an inverse relationship was observed in Experiment 2, it was not significant (P = .101). The skin tensile strength and skin tearing data reaffirm that female broilers have weaker skin than males (Angel et al, 1985; Kafri et al, 1985, 1986; Weinberg et al, 1986; Granot et al, 1991b; Casey et al, 1992; Christensen et al, 1994). Skin tensile strength increased with age in males (Experiment 1) and females (Experiment 2) in concurrence with Kafri et al. (1985) and Weinberg et al (1986) and opposed to an erratic increase reported by Casey et al. (1992) and Christensen et al (1994). Skin strength has been shown to be affected by genetic stain (Kafri et al, 1984; Granot et al, 1991b; Casey et al, 1992). The differences in age-related increases in skin strength observed between Experiment 2 and Christensen et al. (1994) may have occurred because different strains of broiler chickens were utilized. Experiment 1 demonstrated the mitigating effects of L-proline in males by increased skin tensile strength and dermis thickness, and reduced percentage of skin tearing when males were continuously fed halofuginone. The mitigating effect of Lproline in females continuously fed halofuginone was shown in Experiment 2 by increased skin strength and reduced percentage of skin tearing. Experiment 2 also investigated alternatives for continuous dietary inclusion of L-

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proline to mitigate skin quality problems caused by continuous feeding of halofuginone. Glutamic acid is a precursor of Lproline (Shen et al, 1973; Austic, 1976), and many feedstuffs are high in glutamic acid content. Also synthetic L-glutamic acid is less expensive than L-proline. It was hypothesized that if halofuginone increases the requirement for dietary Lproline, perhaps elevated glutamic acid would increase the conversion rate of glutamic acid to L-proline. This hypothesis was not supported because skin strength in the L-glutamic acid treatment was not different from the control at 21 or 42 d of age. At 21 d of age, the diet formulated to provide high levels of L-proline from commonly used feedstuffs had L-proline content similar to the .5% added L-proline diet (1.42 and 1.40%, respectively) but it did not increase skin strength. The differences could be related to the availability of L-proline from natural feedstuffs compared with feeding a synthetic form of Lproline. In Experiment 2, only the treatment providing 1% additional L-proline from 1 to 42 d of age improved skin strength compared with the no added proline treatment at 42 d of age. In Experiment 1, continuous feeding of halofuginone reduced skin collagen levels in agreement with Granot et al. (1991a) and Christensen et al. (1994). Addition of L-proline to halofuginone diets caused significant changes in skin collagen in Experiments 1 and 2, but the changes were not dose-dependent. Addition of .5% Lproline reduced soluble collagen levels of 21-d-old males (P = .018) compared with 0 and 1% L-proline treatments. These observations do not support the theory that halofuginone functions as a L-proline analog. Dermis thickness of males at 21 d of age was increased by .5% added L-proline but 1% did not foster any additional increase. This suggests that .5% added L-proline maximizes dermis thickness and skin strength in male broilers continuously fed halofuginone. This conclusion is supported by the absence of skin tears when .5% L-proline was provided. Dermis thickness was correlated to skin strength, in agreement with Ramshaw et al. (1986). Males continuously fed salinomycin had a

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significantly thicker dermal layer than males fed halofuginone, in agreement with Christensen et al, (1994). Skin tears averaged 7.36% at the processing plant used for these experiments during the previous 2.5 mo when halofuginone was not used. The results suggest that female broilers continuously fed halofuginone would require 1% added dietary L-proline to reduce the percentage of skin tears to levels when halofuginone is not used. The supplementation of L-proline in the diet of male and female broilers increased skin strength, especially in the presence of the anticoccidial drug halofuginone. These experiments suggest that halofuginone's effect on skin strength is more complicated than that of an L-proline analog. Other factors such as the lysine lysl oxidase cross-linking mechanism may be involved. ACKNOWLEDGMENTS The authors acknowledge support from Hoechst-Roussel Agri-Vet Company, Somerville, NJ 08876, and Draper Valley Farms, Mt. Vernon, WA 98273. The diligent work of Charles Nam, Ed Forbus, Ross Hicks, and Pat Walen was appreciated. REFERENCES Angel, S., Z. G. Weinberg, O. Polishuk, M. Heit, I. Plavnik, and I. Bartov, 1985. A connection between dietary coccidiostat and skin tears in female broiler chickens. Poultry Sri. 64:294-296. Association of Official Analytical Chemists, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC. Austic, R. E., 1976. Nutritional and metabolic interrelationships of arginine, glutamic arid, and Lproline in the chicken. Fed. Proc. 35:1914-1916. Blank, T. J., and B. Peterkofsky, 1975. The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts. Arch. Biochem. Biophys. 171: 259-267. Casey, N. H., R. I. Crosley, and G. A. Smith, 1992. Influence of continuous dietary halofuginone on broiler skin tensile strength and growth performance. J. S. Afr. Vet. Assoc. 63:16-19. Casey, N. H., G. A. Smith, and R. I. Crosley, 1989. The influence of breed and sex on the incidence on mortalities and skin tears in broiler chickens. J. S. Afr. Vet. Assoc. 60:102-103.

Christensen, K. D., N. G. Zimmermann, C. L. Wyatt, T. N. Goodman, R. J. Buhr, and P. Twining, 1994. Dietary and environmental factors affecting skin strength in broiler chickens. Poultry Sci. 73:224-235. Crosley, R. I., N. H. Casey, G. A. Smith, and B. Roosendaal, 1992. Influence of phased inclusion of halofuginone on broiler skin tensile strength and growth performance. J. S. Afr. Vet. Assoc. 63:5-11. Granot, I., I. Bartov, I. Plavnik, E. Wax, S. Hurwitz, and M. Pines, 1991a. Increased skin tearing in broilers and reduced collagen synthesis in skin in vivo and in vitro in response to the coccidiostat halofuginone. Poultry Sri. 70:1559-1563. Granot, I., M. Pines, I. Plavnik, E. Wax, S. Hurwitz, and I. Bartov, 1991b. Skin tearing in broilers in relation to skin collagen: effect of sex, strain, and diet. Poultry Sci. 70:1928-1935. Greene, D. E., H. M. Scott, and B. C. Johnson, 1962. The role of proline and certain non-essential amino acids in chick nutrition. Poultry Sci. 41: 116-120. Kafri, I., J. A. Cherry, D. E. Jones, and P. B. Siegel, 1985. Breaking strength and composition of the skin of broiler chicks: Response to dietary calorie-protein ratios. Poultry Sci. 64:2143-2149. Kafri, I., B. S. Jortner, and J. A. Cherry, 1986. Skin breaking strength in broilers: Relationship with skin thickness. Poultry Sci. 65:971-978. Kafri, I., D. J. Zelenka, J. A. Cherry, and P. B. Siegel, 1984. Skin breaking strength in chickens: Comparison among genetic combinations. Poultry Sci. 63:1279-1280. Kao, W. W., R. A. Berg, and D. J. Prockop, 1975. Ascorbate increases the synthesis of procollagen hydroxyproline by cultural fibroblasts from the chick. Biochem. Biophys. Acta 411:202-215. Kuhn, K., 1987. The classical collagens: Types I, n, and IE. Pages 1-42 in: Structure and Function of Collagen Types. R. Mayne and R. E. Burgeson, ed., Academic Press, Inc., New York, NY. Ramshaw, J.A.M., B. J. Rigby, T. W. Mitchell, and A. Nieass, 1986. Changes in the physical and chemical properties of skin collagen from broiler chickens exhibiting the Oily Bird syndrome. Poultry Sci. 65:43-50. Reiser, K., R. J. McCormick, and R. B. Rucker, 1992. Enzymatic and non-enzymatic cross-linking of collagen and elastin. FASEB J. 6:2439-2449. Roy, D. N., and H. R. Bird, 1959. Stimulation of chick growth by proline. Poultry Sci. 38:192-196. SAS Institute, 1987. SAS/STAT® Guide for Personal Computers Version 6 Edition. SAS Institute Inc., Cary, NC. Shen, T. F., H. R. Bird, and M. L. Sunde, 1973. Conversion of glutamic acid to proline in the chick. Poultry Sci. 52:676-682. Smith, T. W., Jr., J. R. Couch, R. L. Garrett, and C. R. Creger, 1977. The effect of sex, dietary energy, meat protein, ascorbic acid and iron on broiler skin collagen. Poultry Sci. 56:1216-1220. Steel, R.G.D., and J. H. Torrie, 1980. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, NY. Stayer, L., 1975. Pages 206-225 in: Biochemistry. W. H. Freeman and Co., San Francisco, CA.

PROLINE AND SKIN STRENGTH IN BROILERS Uitto, J., and D. J. Prockop, 1974. Incorporation of proline analogues into collagen polypeptides. Effects on the production of extracellular procollagen and on the stability of the triple-helical structure of the molecule. Biochem. Biophys. Acta 336:234-251. Weinberg, Z. G., S. Angel, and C. Navrot, 1986. The effects of sex, age, and feed on tensile strength

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of broiler skin. Poultry Sci. 65:903-906. Wilmarth, K. R., and J. R. Froines, 1992. In vitro and in vivo inhibition of lysl oxidase by aminopropionitriles. J. Toxicol. Environ. Health 37:411-423. Zimmermann, N. G., K. D. Christensen, C. L. Wyatt, and P. Twinning, 1994. Effect of halofuginone on broiler skin strength when used in a shuttle anticoccidial program. Poultry Sci. 73:326-330.