Stimulation of Chick Growth with Alfalfa Concentrates

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TAMENESS AND PRODUCTIVITY ACKNOWLEDGMENTS

The authors express appreciation to Drs. J. V. Craig, Department of Poultry Husbandry, and S. Wearden, Department of Statistics, for advice and helpful suggestions on the manuscript, and to Mrs. A. E. Grosse who assisted in making observations on tameness and aggressiveness.

Stimulation of Chick Growth with Alfalfa Concentrates J. A. Liuzzo, J. G. LEE, A. B. WATTS, E . A. FlEGER AND A. F . NOVAK Departments of Agricultural Chemistry and Biochemistry and Poultry Louisiana State University, Baton Rouge

Industry,

(Received for publication September 28, 1959)

N

OVAK et al. (1953) observed that a factor(s) was present in dehydrated alfalfa leaf meal which stimulated growth of the mold, Neurospora sitophila. This factor(s) was distinct from thiamine, folic acid, biotin, para-aminobenzoic acid, vitamin B 12 , riboflavin, niacin, and inositol. Concentration of this factor(s) was reported by Novak, Jonnard and Liuzzo (1958), employing this mold as a test organism for evaluating and comparing the degree of activity in the various fractions. These investigators obtained a final active concentrate which represented less than 0.1% of the solids. This study was proposed to test the growth stimulating effects of these alfalfa concentrates on chicks, since numerous investigations have indicated the presence of an unidentified growth factor(s) in alfalfa for the chick (Kohler and Graham, 1951;

Hansen et al., 1953; Jacobs et al., 1953; Scott et al., 1953; Vavich et al., 1953; Fisher et al, 1954). EXPERIMENTAL PROCEDURE

The growth stimulating effects of concentrates prepared by the method described by Novak, Jonnard and Liuzzo (1958) were tested in three experiments. Figure 1 shows the portion of their fractionation scheme which was used to obtain the concentrates for this investigation. These concentrates were assayed with Neurospora sitophila before each experiment to assess their activity (Novak, Jonnard and Liuzzo, 1958). Growth stimulation ranged from 26 to 45% above the control, indicating highly active concentrates. Vantress White Plymouth Rock broiler cockerels, purchased from a commercial hatchery, were fed their respective rations

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REFERENCES Guhl, A. M., 1953. Social behavior of the domestic fowl. Tech. Bull. 73, Agri. Exper. Sta., Kansas State University, Manhattan. Hediger, H., 1950. Wild Animals in Captivity. London : Butterworths. Hediger, H., 1955. Psychology and Behaviour of

Captive Animals in Zoos and Circuses. New York; Criterion Books. Komai, T., J. V. Craig and S. Wearden, 1959. Heritability and repeatability of social aggressiveness in the domestic chicken. Poultry Sci. 38:356-359. Scott, J. P., and E. Fredericson, 1951. The causes of fighting in mice and rats. Physiol. Zool. 24: 273-309. Siegel, S., 1956. Nonparametric Statistics for the Behavioral Sciences. New York; McGraw-Hill. Snedecor, G. W., 1957. Statistical Methods. Iowa State University Press, Ames. Tindell, D., and J. V. Craig, 1959. Effects of social competition on laying house performance in the chicken. Poultry Sci. 38: 95-105. Wood-Gush, D. G. M., 1955. The behaviour of the domestic chicken: A review of the literature. Brit. J. Anim. Behav. 3 : 81-110.

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J. A. Liuzzo, J. G. LEE, A. B. WATTS, E. A. FIEGER AND A. F. NOVAK Dehydrated

alfalfa leaf

meal

extracted with O.I N HCl i

'

Residue (discarded) 1

1

Extract . . . . . precipitated with 9 5 % ethanol

1

Filtrate

Precipitate

Aqueous phase

\ \

Extract

IICOIKJMI. o

' C o n c e n t r a t e A)

rt

extracted with chloroform

Aqueous phase (Concentrate

/ /

Extract

B)

FIG. 1. The portion of the fractionation scheme, proposed by Novak, Jonnard and Liuzzo (1958), which was employed in this investigation.

ad libitum for eight weeks. They were weighed weekly and feed consumption data were collected. Experiment 1 was conducted during the months of August and September; experiment 2 in November and December; and experiment 3 in June and July. The first two experiments were conducted in a different building from the one used for experiment 3. This allowed observations to be made under different environmental conditions. Experiment 1. Forty day-old chicks were allotted at random into eight groups. Four treatments were employed, each consisting of two groups. Treatment replicates were housed in separate pens and arranged in wire floored batteries at different pen levels. The chicks remained in starter batteries for five weeks at which time they were moved to wire floored finishing batteries. The four treatments consisted of the basal, basal + 5% alfalfa leaf meal (17% protein), basal + HCl extract, and basal + concentrate A. The HCl extract and concentrate A were supplemented to the

TABLE 1.—Composition of the basal ration Ingredients Yellow corn meal Soybean oil meal Menhaden fish meal Dicalcium phosphate Oyster shell flour Iodized salt Animal fat Vitamin Premix 1

% 61.5 27.4 3.0 2.0 1.0 0.5 4.0 0.6

1 Supplies the following per lb. of ration: vitamin A 5,000 I.TJ., vitamin D 8 640 i.c.tr., vitamin E 2.4 I.u., menadione 4 mg., riboflavin 2 mg., calcium pantothenate 4 mg., niacin 9 mg., vitamin Bi2 .006 mg., choline chloride 500 mg., bacitracin 5 mg., manganese 40 p.p.m., and methionine 23 gms.

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extracted with diethyl ether

basal at a level equivalent to 5% alfalfa. The composition of the basal ration is shown in Table 1. When alfalfa meal was added to the basal, the corn meal was reduced proportionately. Experiment 2. Two hundred day-old chicks were randomized into groups of 20. Five treatments were employed, each consisting of two replicates or 40 chicks per treatment. Housing allotments were the same as in the first experiment. The treatments consisted of the basal, basal + 3 % alfalfa leaf meal, basal + HCl extract of alfalfa leaf meal, basal + concentrate B (Figure 1), and basal + concentrate C (combination of ethanol precipitate, and ether and chloroform extracts). All extracts and concentrates were supplemented at a level equivalent to 5% alfalfa. Experiment 3. Two hundred day-old chicks were placed on the Poultry Department's farm ration for five days. This ration consisted of the ingredients shown in Table 1 plus 3 % dehydrated alfalfa leaf meal (corn meal level adjusted, proportionately). On the sixth day, the chicks were allotted according to weights into the same design used in experiment 2. This weight allocation was employed to reduce the amount of variability between lots, which existed in the previous experiments.

825

ALFALFA CONCENTRATES AND GROWTH

RESULTS AND DISCUSSION

The weight gains shown in Table 2 indicate that a growth stimulating substance (s) was present in the HC1 extract and in the concentrates. In experiment 1 the HC1 extract displayed its greatest stimulation over the basal at the third week, whereas concentrate A showed its maximum at the fifth week (6.5 and 12.7% above the control, respectively). In experiment 2 maximum stimulation for the HC1 extract and concentrate B occurred at two weeks (8.8 and 5.9% respectively). In contrast to these experiments, the highest response in experiment 3 occurred at eight weeks for all supplemented rations. The TABLE 2.--Weekly

ration supplemented with concentrate B showed the largest response (16.7%). Those containing the alfalfa, HC1 extract, and concentrate C resulted in less stimulation (10.5, 11.7, and 11.3%, respectively). These results indicate that maximum response from this growth substance (s) may occur at any time during the eight week period. An analysis of variance of the weight gains at eight weeks for chicks in experiment 1 showed the treatment differences to be statistically significant at the 1 % level. The studentized range (Duncan, 1955) indicated the following differences: (at the 1% level of significance) basal + concentrate A and basal + 5 % alfalfa, and basal + concentrate A and basal; (at the 5% level) basal and basal + HC1 extract, and basal + HC1 extract and basal + 5% alfalfa. It is apparent from the results in Table 2 that under the conditions of experiment 1, supplementation of alfalfa leaf meal at the 5% level resulted in significantly lower gains than were evident for the chicks receiving the extract and concentrate. Because the question of maximum supplemenweight gain averages1 Week

Experiment

Ration

1

1

2

3

4

5

6

7

8

Basal B a s a l + 5 % alfalfa Basal+HCl extract Basal+concentrate A

.16 .14 .16 .16

.43 .38 .45 .43

.77 .70 .82 .80

1.12 1.05 1.17 1.20

1.42 1.40 1.50 1.60

1.93 1.86 2.03 2.12

2.37 2.32 2.52 2.62

2.87 2.82 3.05 3.20

2

Basal B a s a l + 3 % alfalfa Basal+HCl extract Basal+concentrate B Basal+concentrate C

.14 .14 .14 .14 .13

.34 .35 .37 .36 .33

.64 .67 .67 .67 .62

1.01 1.03 1.04 1.05 0.98

1.44 1.45 1.47 1.48 1.39

1.91 1.93 1.93 1.96 1.86

2.59 2.63 2.61 2.62 2.56

2.88 2.92 2.93 2.95 2.88

3

Basal B a s a l + 3 % alfalfa Basal+HCl extract Basal+concentrate B Basal+concentrate C

.14 .17 .14 .13 .15

.38 .39 .38 .39 .42

.60 .64 .65 .65 .67

0.89 0.96 0.97 1.00 0.98

1.22 1.28 1.29 1.32 1.34

1.56 1.71 1.69 1.79 1.70

1.99 2.19 2.21 2.32 2.21

2.39 2.64 2.67 2.79 2.66

1

Expressed in lbs.

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The chicks were housed in the same starting and finishing batteries that were used for the preceding experiments. A fruit press was employed to extract the HC1 solution from the alfalfa residue after autoclaving. Consequently, the separation was more complete in this experiment than in the previous two, where gravitational filtration was used. The treatments and levels of supplementation were identical with those of experiment 2.

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J. A. Liuzzo, J. G. LEE, A. B. WATTS, E. A. FIEGER AND A. F. NOVAK

TABLE 3.—Feed conversion averages'Experiments Ration Basal B a s a l + 5 % alfalfa B a s a l + 3 % alfalfa Basal+HCl extract Basal+concentrate A Basal+concentrate B Basal+concentrate C 1

1

2

3

2.08 2.03

2.18

2.20

•

—

2.06 2.04

— —

Lbs. of feed per lb. of gain.

—

2.17 2.16

—

2.16 2.17

—

2.17 2.13

—

2.06 2.18

plemented with such crude materials as fish solubles and condensed distillers solubles as well as with pyridoxine, biotin, folic acid, copper, cobalt, potassium, iron, molybdenum and sulfur (as the sulfate). Although the basal gains in experiment 3 were not as high as in the first two experiments, they are considered to be normal for chick investigations conducted at this station during the hot summer months. The chicks receiving the extracts and concentrates displayed the best feed conversion (Table 3). However, these differences were not significant. It is interesting to note the growth stimulation caused by concentrate C in the third experiment. This growth promotion which failed to occur in experiment 2, could be attributed to an increased recovery of solubles due to the use of a fruit press for separating the HC1 extract and alfalfa residue. It is not at all unusual to find activity divided between such fractions. However, it is possible that an ethanol insoluble growth factor(s) was extracted by the more complete separation. In experiment 2, recovery of this substance (s) may have been too minute to display a stimulatory effect, since separation was performed by gravitational filtration. Results of these experiments show that the concentrates prepared from alfalfa leaf meal by Novak, Jonnard and Liuzzo (19S8) which were active for Neurospora sitophila are required also for maximum growth of chicks. These may contain the same chick growth factor(s) which other investigators have indicated to be present in alfalfa. SUMMARY

Three chick experiments were conducted to test the growth stimulating effects of concentrates prepared from dehydrated alfalfa leaf meal. The concentrates, previ-

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tation of alfalfa for optimum results in different rations is still debatable, no explanation is offered for the lower gains made by the chicks on this ration. To compensate for this effect, the level of alfalfa meal was lowered to 3 % in the subsequent experiments. In experiment 2, after the fourth week, the degree of response above the control decreased considerably for all supplemented rations. This reduction was less dramatic in the first experiment. Because of this tapering effect, weight gains were analyzed at the fourth week. An analysis of variance indicated a treatment difference, significant at the 5% level. The studentized range showed differences existed (5% level) between the basal and basal + concentrate B, and the basal + concentrate C and all other rations except the basal. An analysis of variance of the weight gain data in experiment 3 indicate a treatment difference, significant at the 1% level. The studentized range showed that the basal gains were significantly ( 1 % level) lower than the gains of chicks receiving the supplements. The differences which existed between the basal and basal + concentrates are extremely important, since this practical ration has been used by the Louisiana Station for a number of years and has failed to show significant improvement when SUP-

ALFALFA CONCENTRATES AND GROWTH REFERENCES

Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 1 1 : 1-42. Fisher, H., H. M. Scott and R. G. Hansen, 1954. Further studies on the alfalfa factor and its relation to the liver and whey factors. J. Nutrition, 52: 13-24. Hansen, R. G., H. M. Scott, B. L. Larson, T. S. Nelson and P. Krichevsky, 1953. Growth stimulatory and growth inhibition of chicks fed forage and forage juice concentrate. J. Nutrition, 49: 453-463. Jacobs, R. L., J. F. Elam, J. H. Quisenberry and J. R. Couch, 1953. Dehydrated alfalfa leaf meal as a source of vitamins and unidentified factors for the mature fowl. Poultry Sci. 32: 812-816. Kohler, G. O., and W. R. Graham, 1951. A chick growth factor found in leafy green vegetation. Poultry Sci. 30:484-491. Novak, A. F., A. R. Gama, J. A. Liuzzo and E. B. Rubloff, 1953. Factors affecting the microbiological assay of pyridoxine in multivitamin products. J. Am. Pharm. Assoc. 42: 581-583. Novak, A. F., M. L. Jonnard and J. A. Liuzzo, 1958. Concentration of an alfalfa growth factor for Neurospora sitophila and its use in the microbiological assay of pyridoxine. J. Am. Pharm. Assoc. 47: 413-416. Scott, H. M., H. Fisher and J. M. Snyder, 1953. Alfalfa meal as a source of unidentified growth factors. Poultry Sci. 32: 555-556. Vavich, M. G., A. Wertz and A. R. Kemmerer, 1953. Growth-stimulating factors in alfalfa for chicks. Poultry Sci. 32: 433-436.

Observations on Hatching Time in Three Avian Species URSULA K. ABBOTT AND ROBERT M. CRAIG Department of Poultry Husbandry, University of California, Davis, California (Received for publication September 28, 1959)

F

OR high hatchability and good poult or chick quality, the majority of embryos should be at similar stages of development when they are transferred to hatching conditions. Ideally all poults or chicks should pip the shell and hatch within the same period. Poor results are obtained when embryos of different developmental

age are expected to hatch together or when variation in pipping time is high. In extreme cases, i.e., eggs from highly inbred stocks or eggs that have been held for long periods prior to setting, hatching may continue over four or more days. Under such circumstances the earliest hatched poults or chicks are weak and dehydrated before

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ously found to be active for Neurospora sitophila were prepared according to the method of Novak, Jonnard and Liuzzo (1958), and involved acid-extraction of alfalfa leaf meal, precipitation of the extract with ethanol, extraction of the ethanol filtrate with (1) ether (concentrate A) and (2) both ether and chloroform (concentrate B). Concentrate C was a combination of the ethanol precipitate, and the ether and chloroform extracts. The concentrates were supplemented to the practical ration routinely used by the Louisiana Station at a level equivalent to 5% alfalfa leaf meal. The chicks were fed these rations ad libitum for eight weeks. Significant growth responses above this practical control were obtained in the first experiment with concentrate A. Therefore, fractions B and C were prepared and both were tested in the second and third experiments. Significant growth responses were obtained in the second experiment with concentrate B; and in the third experiment with concentrates B and C. These results show that the concentrates prepared from alfalfa leaf meal by Novak, Jonnard and Liuzzo (1958) which were active for Neurospora sitophila are required also for maximum growth of chicks.

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