SPARING ACTION BETWEEN METHTONTNE, CHOLINE AI\ID VITAMIN B.I2 IN CHICKS AND HENS SLINGER, W. F. PEPPER and J. D. SUMMERS Departments of Nutrition and Poultry Science, Unive,"sity of Guelph, Guelph, Ontario, Canada. Received May 6, 1970, accepted July 29, 1970. S. J.

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ABSIRACT Two experiments were conducted with White I-eghord strain-cross chicks and two with the sarie strain of hens in an attempt to determine whether the methionine supplement frequently added to practical coin, soybean rireal-type diets coufd be partially or com-replaced

B,, spared ttre chicks' qeeds for methionine. Based on the statistical analysis, the-laying diets were adequate in methyl groups for ,egg

production and feed efficien-cy, _since choline and vitamin 8,, w-ere not effective in sparing the requiremeni of methionine for these functions. On the other hand, in- one. experiment choline supplementation resulted i1 slight iT-

pletely by cholini and/br vitamin -Br. Methionine, chbtne and vitamin 8,, were st;died using i factorial design. Sigiificant provemenf in several parameters of economic growth and-feed efficiency responsis with importance, suggesting,a need for additional chicks were obtained only-with-methionine, methyl groups in the diet gmployed, Vitam-in suggesting that the diet was adequate in B,, was found to be capable of sparing the methyl gioups as well as in essentiaf choline neEd for methionine for egg size, under cerand vitamin B-. Neither choline nor vitamin tain circumstances.

INTRODUCTION

Quillin et al. (9) and Leach et aI. (5) have reviewed the literature on the interrelationships between methionine, choline and vitamin B. in chicks and hens. The evidence indicates that both methionine and choline furnish labile methyl groups as well as assuming independent roles in nutrition. For methylation, choline is oxidized to betaine which serves as the active source of methyl goups. Betaine and methionine can replace the dietary need for choline as a source of labile methyl groups, but choline per se is needed by chicks for the prevention of perosis. The need for methyl groups is influenced by the level of vitamin Bo and folic acid, since these vitamins are known to function in the metabolism of l-carbon fragments. In the chick experiments of Quillin et al. (9), three levels of choline and three or four levels of methionine were added in all possible combinations to practical corn, soybean meal rations containing lO or l27o fat. The addition of methionine produced a significant growth response only with low-choline rations, while the improvement in growth which accompanied the addition of choline approached statistical significance only with low-methionine rations. The addition of both was not significantly better than the addition of either one alone at the highest levels used. Based on the results obtained, 1 g of choline was calculated to be equivalent to approximately 2.3 to 2.4 g of. Dl-methionine as a methyl donor in these rations. With hens, Johnson (4) found little or no improvement in egg production upon the addition of vitamin B* or choline to a corn, soybean-type diet. As a matter of fact, the results raised the question as to whether supplemental choline (500 and 1000 mg/kg of diet) may actually reduce egg production and hatchability. Hoknes and Kramer (3) reported that the addition of choline, without supplementary methionine, resulted in a significant decrease in egg production when egg-type pullets were fed a diet in which soybean meal was the sole protein concentrate. The addition of vitamin B- or methionine resulted in an improvement in egg production. Adding high levels of choline or vitamin Bo had an Can. J. Antm. Sci. 50: 693-703 (Dec. 1970)

693

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694

cANADTAN

JouRNAL oF ANTMAL scrENcE

adverse efiect on the efficiency of feed utilization, except in the presence of supplemental methionine. In more recent work, Grffith et al. (2) found that choline, methionine or yifamin Bo increased egg production and egg size when added to diets high in corn and soybean meal. Supplementation of the diet with choline reduced liver fat levels, while methionine and vitamin B- had little or no effect on liver fat. The diet which was supplemented with all tlree nutrients caused grcater increases in egg production and egg size and a greater decrease in liver fat than any one

nutrient added alone. The present report concerns experiments designed to determine whether the methionine supplement frequenfly added to practical corn, soybean-type rations for egg-strain chicks and hens may be partially or completely replaced by the less expensive supplements of choline and/or vitamin B., and whether supplements of these factors, alone or in combination, may be beneficial.

MATERIAIS AI\[D METIIODS Experiments 1 and 2

These experiments involved the use of White Leghorn strain-cross (Shaver Starcross 288) chicks. Experiment I comprised four replicate groups of 21 female chicks per treatment. Experiment 2 was carried out at a difierent time and comprised six replicates of 10 male chicks per treatment. Both experiments were of the same randomized block design and comprised a 2 x 2 x 2 factoial axrangement with two levels of added Dl-methionine (0 and o.o|Vo ), two levels of added vitamin B* (0 and 13.2 pe/kg and two levels of added choline (0 and 330 mglkg). The chicks were distributed at random in electrically heated, wire-floored pens at one day of age. The appropriate experimental diet and water were supplied ad libitum for a four-week period. Weight change, feed const'mption and mortality data were collected. All supplements were premixed with ground yellow

corn prior to inclusion in the diets. The premix additions were made at the expense of ground corn. The basal diet (Table 1) was assayed for protein (N x 6.25) using a macro-Kjelda}l procedure, and for methionine and cystine using a microbiological method (11). The choline and betaine contents of the basal diet were determined using the reineckate method of Glick (1), and vitamin B* was assayed by the U.S.P. microbiological method (12). The data resulting from the experiments were subjected to statistical analysis using the method of analysis of variance (10), and the major treatment efiects were broken down to individual degrees of freedom. Experiments 3 and 4

The experiments were conducted using White Leghorn, strain-cross (Shaver Starcross 288) pullets at point of lay. Both experiments were factorial arrangements set up in a randomized block design. Experiment 3 comprised two levels of protein (13.5 and 15.5% ), two levels of added methionine (0 and O.O5%), two levels of added vitamin B. (0 and 4.95 pg/r;g and two levels of added choline (0 and 330 mg/kg). Three replicate groups of 11 females, 24 weeks of age, were placed in each experimental treatment. All birds were housed individuatly in 20-cm wire cages. Feed and water were supplied ad libitum. To the basal diet (Table 1), supplements were added as described for experiments L and 2.

SLINGBR ET AL._SPARING ACTION OF SUPPLEMENTS

Table

l.

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Ingredient Ground yellow corn Wheat middlings Ground wheat Soybean oil meal (50/e protein) Meat meal (50/e protein) Dried whey (65/6 lactose) Dehydrated alfalfa meal (17/6 protein) Animal tallow (stabilized) Dicalcium phosphate Ground limestone Iodized salt (0.015% KI) Vitamin: mineral premix

Vitamin: mineral premix

Vitamin A (10,000 IU/g) Vitamin D3 (1,650 ICU/g)

d-Calcium pantothenate (70.5 mg/g) Riboflavin (52.9 mS/S) Niacin Menadione sodium bisulfite Zinc oxide (807o Zn) Manganous oxide (5616 Mn) Copper sulfate (25le Cu) Ground yellow corn

Total (g)

695

Composition of basal diets Experiments

Experiments

% 46.0

6r.25

land2 5.0 34.0 2.5 2.0 2.0 5.0 1.5

1.0 0.5 0.5

3and4 d

/o

10.0 17.0 2.O

1.5 2.25

5.5 0 .25

o.25

9/100 kg/diet

9/100 kg,zdiet

50.0 25.0 6.2 8.4

s7 .4

62.S

6.4

1.1 12.5

tz.s

6.4

6.4

12.5

12.5

3.3

377 .9

r2l

500 .0

250.0

.5

Analvsis

Protein (N X 6.25), % Metabolizable energy (kcal/kg) Choline (-glkg) Betaine (mglkg)

Vitamin Bt, (ttT/kr) Methionine (/6) Cystine (/p)

24.8

15.4

3040

2880

1

718

t245

251

163

3.1

8.4 o.27 o.20

0.36 0.36

The 13.5% protein diet was made from the l5.5Vo protein basal by increasing the amount of corn and lowering the amount of soybean meal; no attempt was made to maintain a constant energy level. Records were kept on total feed consumption, total egg production, body weight changes and mortality for the 10 28-day periods of the experiment. Three days' eggs were saved during each 28-day period in order to determine average egg weight. Experiment 4 was an additional hen experiment conducted in the sarne facilities

one year later. This factorial involved two levels of added methionine (0 and O.OSVo ), and three levels of added choline (0, 330 and 660 mgtkg. The same 15.5% protein basal diet as employed in the previous experiment was used. The basal diet contained no added vitamin Bo but an additional treatment falling outside the factorial and involving supplementation of the basal with 6.6 pg/kg of vitamin

B.

was included.

The strain of pullet, the housing conditions, the number of birds per replication and per treatment, and the length of the experiment were the same as for

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696

cANADIAN JoURNAL

oF ANIMAL scIENcE

experiment 3. The same criteria of measurement as used in that experiment were also used, and the following additional data were collected: egg specific gravity, percent weight loss after incubating the eggs for seven days, Haugh units and blood spots determined on three days' eggs for each of the 10 28-day periods. At the conclusion of the experiment all birds were sacrificed and the livers were removed for fat determinations which were conducted on pooled liver samples from each replication. Protein, choline, vitamin Bo, betaine, methionine and cystine were determined as indicated for experiments L and 2. Fat in the livers was determined by extracting a sample of the lyophilized liver in a soxhlet apparatus for 16 hours with

petroleum ether. All data were analyzed statistically by the method of analysis of variance, and the major treatment effects were broken down to individual degrees of freedom

for

comparison.

Experiments

RESULTS

I anil 2

The results of these experiments are presented in Table 2. The addition of methionine produced a significant (P < 0.01) improvement in both weight gain and feed efficiency, in both females (experiment 1) and males (experiment 2). Neither vitamin B- ror choline supplementation resulted in significant changes in weight gain or feed efficiency in either experiment. Mortality was low for both experiments and deaths were not related to any particular dietary treatments. Experiments 3 and 4 Statistical analysis of the data for experiment 3 (Table 3) revealed no significant differences in average egg production, feed intake, feed per dozen eggs or body weight change for any of the treatments. Overall means for egg production and kg feed per dozen eggs for birds fed the 13.5 and. 15.5% protein diets were 75.8 and,77.4Vo, and 1.70 and 1.67, respectively. Means for 0 and O.OSVo added methionine werc 76.2 and 77.OVo, and 1.69 and 1.68, respectively, for egg production and kg feed per dozen eggs. Means for 0 and 4.95 pgkg of vitamin Bwerc 77.1 and 76.0, and 1.67 and 1.70 for percent egg production and kg feed per dozen eggs. The overall means for 0 and 330 mg/kg of added choline were 76.7 and 76.5, and'1..67 and 1.69, respectively. Table 2. Effect of methionine, choline and vitamin Br2 supplements on weight gain and feed efficiency in strain-cross, White Leghorn-type chicks Experiment 1, females Experiment 2, males

Supplements added*

DL-

Gainlchick

Vitamin

methionine Brr

(g) 26e (32)I

a 1A

1

26+ (84) 270 (83) 272 (8s) 283 (83) 286 (83) 280 (83) 282 (8s)

2.25

I

)

-r

J

4

(

-r

6

-l-

8

1 + +

+

*Dl-methionine added at rate of

Feed: gain

Choline

I 1 +

2.22

2.r8

2.tl

2.09 2.15 2.17

Gainlchick (g)

2e0 (60)

287 (se) 283 (60) 2e1 (60) 301 (60)

2e8 (se) 302 (60) 2es (se)

Feed: gain

2.04 2.07 2.08 2.07 2.O0

2.01

2.00 2.O2

.2 pe/kc, and choline at rate of 330 mglkg of diet. fFigures in parentheses represent numbers of chicks remaining at conclusion of *periment, O

.OSVo,

vitanin Brr at rate of

13

SLINGER ET'AL.-SPARING ACTION OF

Table

3. Effect of choline,

Methionine Vit.

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(%)

added

I'c/ks)

Choline added

(mglkg)

3.5

J..') .1..)

3.5 J.J

5.5 5.5 5.5 5.5 J.J

5.5

+ls 4 .95

ols

330 330

+ss

4.95

330 330

495 o 0 0 0

-os .05 4 .95

.05 .05

4.95

(kg)

7s.7 .4

107

.66

74.8

106

.7r

74.7

107

.72

75.r

105 105 108

.69 .65 .64

107

.,J

330

79 .O

330

74.r

r07

330

78.9 80.3

108 109 106 108

74.7

gso

eggs

.73 .69 .64

76.9

4.95

doz.

104 109 108 108

77

0 .05 0 .05 0 .05

Feed/

' 77 .4 79 .r

L .J

3.5 3.5

J.5

egg Feed/ orod. bird/ H.D.B.* day (g) (%) Av.

Br2

added

(%)

697

methionine and vitamin Br: on laying hen performance, experiment 3

Treatment Protein

SUPPLEMENTS

79 .7

75.3

.72

,7A

;65

.63 .60 .72

Av. egg wt. (g)

55.5 56.7 55.9 57.0 57 .2 58.9 57 .9 56.4 56.5 58.1 59.0 58.8 59.0 58.3 58.2 58.9

Av.

wf.

gain (g) 249 233 279

288 289 299 274 336

254 315

438 302 345 292 365

i72

+Hen day basis.

The analysis revealed a highly significant (P < 0.01) improvement in egg weight with the higher level of protein (57.0 vs. 58.4 g). A higNy significant response in egg weight was also noted upon the addition of O.OSVo Dl-methionine (57.2 vs.58.1 g). The methionine x vitamin B* interaction was significant for average egg weight (Table 4). Supplementary vitamin B- increased egg weight in the absence and decreased it in the presence of added methionine, while added methionine increased egg weight in the absence but not in the presence of supplementary vitamin B*. The only significant responses noted in experiment 4 (Tables 5, 6, 7 and 8) were alinear (P < 0.05) decrease in egg specific gravity with increasing levels of choline (1.080, 1.O79 and I.O77) and a quadratic (P < 0.05) interaction for methionine x choline based onHaugh units (Table 8). The methionine x choline interaction is shown in Table 9. The 330 mC/4C level of added choline decreased Haugh units in the absence of added methionine and increased this measurement in the presence of added methionine. The addition of methionine appeared to decrease Haugh units on the lowest and highest levels of choline, and to increase Haugh units at the intermediate level of choline. Table

4. Interaction of methionine

and vitamin B12 on egg

weight, exPeriment

3

Egg weight (g) Methionine added (To)

Vitamin Br: added

0 .05

fus/ks) 0

4.95

JO. /

58.4

Jl.t

57 .4

CANADIAN JOURNAL OF ANIMAL SCIENCE

698

5.

Table

Effect of choline, methionine and vitamin Brz on laying hen performance, experiment 4

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Treatment

Av. wt. gain (g)

ess Feed/ Feed/ doz. eggs (kg) (%) (g)

Av.

prodl" bird/ Choline Vitamin added Brz added H.D.B.* day

Methionine

(o'slkg) (t's/ks)

added (%)

00 330 660 00 330 660 0

0 0 0

0.05 0.05 0 .05

ot

77 .9 77 .3

0 0

76.r

76.7

78.4

0

79.5

0

6.6

77

.r

109

.68

109 108 107 108 110 110

.69 .70

Liver fat dry wt. basis

(%)

393 433

48.8

383

44.3

-o/

JJ'

.65 .66

403

39 .8 47 .5 43 .8

AJ'

42.4

462

48 .0

.7t

xHen day basis. fNot included in statistical malysis.

Table

6. Overall effects of methionine and choline on laying

hen performance and egg weight,

exPeriment 4

Av. egg

production H.D.B.*

Treatment Methionine added (To) 0

Choline added (mg/kg) 0 330 660

bird/

egg

Liver fat dry wt.

weight

basis

doz.

day (g)

(kg)

(g)

(%)

108

| .70

55.6

80.7

r07

1.65

56. 3

44.3 44.6

78.6 80.7

r07

1 1

55.6

48.1

109

80.4

110

1.67

56 .3 56 .0

4l .l

79

0 .05

Av.

Feed/

Feed/

.r

.70 .65

44.O

*Hen day basis,

Table 7. Effect of choline, methionine and vitamin Br: on eBg characteristics, experiment Treatment Methionine added

Choline added

(%)

(mglkg)

0 0 0

0 330

0.05 0.05 0.05 0+

660 0 330

660 0

*Not included in statistical analysis.

Vitamin Av. egg Av. egg Av. egg Brz added weight specilic weight (g) gravity loss(lo) Q.rc/kd 0

56.8

0 0 0 0 0

57 .2

56.8 57.9

6.6

56.9

56.7 58.2

.080 .080 .077 .080

.o79 .o77 .080

Blood spots

Av. Haugh

units

9.7

74.5 7r .9

9.3

7r .8

10.1

9.9

9.4 9.8

9.1

/.)

.

.)

7r.9 70.3

Large

(%)

Small

3.2 2.9 3.1 2.7 3.6 2.8 6.0

4.7 3.9

(%)

t.7

4.6

4-3 4.6 3

.8

4

699

SLINGER ET AL.-SPARING ACTION OF- SUPPLEMENTS

Table 8. Analysis of variance for specilic gravity and Haugh unit data Specific

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Source of variation

Methionine Choline (L) Choline (Q) Methionine X choline (L) Methionine X choline (Q)

Error

*Differencessignificant (P fwith 10 d.f. for error.