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Measurement and Differentiation of Unidentified Chick Growth Factors Using a New Semi-Purified Ration
Measurement and Differentiation of Unidentified Chick Growth Factors Using a New Semi-Purified Ration R. A. RASMUSSEN,* PAUL W. LUTHY, J. M. VAN LANEN AND C. S. BOROTF Hiram Walker &• Sons, Inc., Peoria, Illinois (Received for publication July 9, 1956)
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* Present address: Peter Hand Foundation, Chicago 22, Illinois.
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tors. Fisher et al. (1954) postulate three distinct chick growth factors present in alfalfa meal, dried whey, and defatted whole liver powder, respectively. A further report by Scott (1955) from the same laboratory adds a fourth factor found in distillers dried solubles. Rasmussen et al. (1954) reported the presence of two unidentified chick growth factors in distillers dried solubles. One factor is "vitamin Bi3" and the other produces responses of about the same magnitude as those secured from liver residue. Combs et al. (1954) found the chick to require three unidentified growth factors—one found in condensed fish solubles and liver fraction NF 2 (Armour and Company); the second in dried whey products, dried molasses distillers solubles, and dried brewers yeast; and the third in dehydrated alfalfa meal and dried brewers yeast. Peterson et al. (1955) reported a factor common to herring fish meal, sardine fish solubles, 50% dried lactose whey product, BioPar "C," and defatted liver residue. All of these items produced equal but not additive growth responses when added to their basal purified ration. Arscott and Combs (1955) noted that condensed fish solubles contains two unidentified factors, one of which is present in a liver fraction and the other in dehydrated alfalfa meal. March et al. (1955) elucidated two unidentified chick growth factors in dehydrated cereal grass and herring meal. The response to either was dependent on the presence of the other in the ration.
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HE presence of unidentified nutritional factors in various ingredients fed to chicks has been well established. Much of the literature on this subject has been reviewed by Menge et al. (1952). Considerable evidence has accumulated pointing to a multiplicity of unidentified factors required by the chick. Carlson et al. (1949) postulated the presence of two distinct factors, one in fish meal and the other in dried brewers yeast. Stokstad and Jukes (1950) reported that at least part of the chick response to Streptomyces aureofaciens materials was due to chlortetracycline. Kohler and Graham (1951) concluded that the unidentified forage crops juice factor is different from vitamin B13, whey factor, or antibiotic effect. Couch et al. (1952) found one unidentified chick factor in distillers dried solubles, dried whey, and whey product and another in fish meal. Edwards et al. (1953) reported that fish solubles and penicillin mycelium residue contain the same unidentified chick growth factor(s). Menge et al. (1952) found that two distinct unidentified factors are required by the chick for rapid growth. One of these is present in liver fraction "L," liver fraction BioPar " C , " and dried brewers yeast, and the other in dried whey or dried whey products. Norris (1954) reported that fish solubles, distillers dried solubles, and grass juice each contain distinct unidentified growth fac-
UNIDENTIFIED GROWTH FACTORS
EXPERIMENTAL PROCEDURE
New Hampshire X Indian River or Columbian crossbred chicks were used in all experiments. These chicks were progeny of dams kept in individual-pen laying batteries with raised wire floors. During the period in which eggs were collected for incubation the hens were artificially inseminated twice weekly with fresh, pooled semen from males kept in individual-pen batteries with raised wire floors. Little was gained by depletion of the hens through feeding an all vegetableprotein diet. Both controls and supplemented chicks from depleted hens were correspondingly smaller at four weeks than chicks from hens fed rations con-
taining animal proteins. Therefore, the breeder diet for these studies was fortified with unidentified factor carriers. The breeder diet had the following percentage composition: 65.75 yellow corn, 14 Hi-Pro-Con (50% protein soybean oil meal), 3 meat and bone scraps, 2 fish meal, 2 dried whey, 3 distillers dried solubles, 3 alfalfa meal, 0.2 viadex (4,000A750D), 3 dicalcium phosphate, 3.5 limestone, 0.5 trace mineral salt, 0.0066 manganese sulfate, 0.025 diphenyl-pphenylenediamine, 0.025 riboflavin supplement (8,000 mcg./gm.), 0.00147 niacin, and 0.00088 calcium pantothenate. Oyster shell was available ad libitum. The basal chick diet had the following percentage composition: 56.18 Cerelose, 0.25 corn oil, 17.5 blood meal, 14 Hi-ProCon, 3.5 gelatin (Keystone No. 11 edible), 2 Stamino type A (A. E. Staley Company containing 10% isoleucine), 0.3 methionine, 1.1 vitamin mixture, 0.5% trace mineralized salt (Hardy formula #2) and 4.67 minerals. This basal contained 27.8 percent protein and 1,077 Calories of productive energy (P.E.) per pound (Titus, 1955). By calculation all essential amino acids not specifically tested and found adequate were present in excess of NRC requirements adjusted for a ration of 27.8% protein. The vitamin mixture furnished per kilogram of ration the following: 6,040 I.U. vitamin A, 1,130 I.U. vitamin D3, 2 gm. choline chloride, 0.2 mg. vitamin Bi2, 20 mg. thiamine, 7 mg. riboflavin, 20 mg. calcium pantothenate, 32 mg. niacin, 7 mg. pyridoxin, 0.2 mg. biotin, 1.4 mg. folic acid, 1 mg. Klotogen F (sodium bisulfite salt of Menadione), 2 mg. paminobenzoic acid, 100 mg. inositol, 20 mg. alpha-tocopherol acetate, 100 mg. ascorbic acid, and 250 mg. diphenyl-pphenylenediamine. With 4% dicalcium phosphate, 0.6%
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Norris (1955) and Couch et al. (1955) reported that at least part of the unidentified factor response secured from distillers dried solubles and other unidentified factor supplements is obtained from the ash of these supplements. Benton et al. (1955a,b) indicated that half of the growth activity of gelatin was due to an ash component. Combs (1955) reported that, under his conditions, distillers solubles ash failed to produce a response but that a combination of ash from fish solubles, dried whey and alfalfa meal promoted chick growth slightly. Scott el al. (1955) secured a significant chick growth response to distillers solubles but not to distillers solubles ash. Fisher et al. (1954) found an unidentified factor in alfalfa not associated with alfalfa ash. This paper presents results of studies conducted since June of 1953 on the unidentified factors required for chick growth employing a relatively inexpensive semipurified diet which produces good growth. At least three different unidentified factors are evident with this ration.
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48
R. A. RASMUSSEN, P. W. LUTHY, J. M. VAN LANEN AND C. S. BORUFF
Additions to the basal ration were made at the expense of Cerelose. RESULTS
Growth responses with graded levels of alfalfa and whey are shown in Table 1. These results reveal no additive effect of alfalfa (A) and dried whey (W). The alfalfa meal (17 percent protein, artificially dried) did not equal dried whey at equivalent levels nor did it sharply depress growth at the higher levels as observed by others, (Lepkovsky et al., 1950; and Kodras et al, 1951a, b). However, continued trials with these two supplements singly and in combination suggests that the same factor is supplied by both but that alfalfa reaches a lower maximum response because of its "inhibitor" content. In some trials 10 percent alfalfa gave a definite growth depression. The very similar responses from alfalfa and whey and from whey alone indicate a single factor. In subsequent experiments, 5 percent alfalfa and 5 percent whey which gave near maximum response were employed as a single factor source (AW). Other investigators (Fisher el al., 1954; and Combs et al., 1954) observed a growth differential between alfalfa meal and dried whey pointing to the existence TABLE 1.—Growth responses of chicks to alfalfa and whey supplementation Av. 4-wk. wt.* (gms.)
% gain over controls
Feed/Wt. gain
None
354
-
2.6
5 . 0 % Alfalfa Meal (A) 7.5% A • 10.0% A
422 411 422
19 16 19
2.5 2.4 2.3
5 . 0 % Dried Whey (W) 7.5% W 10.0% W
455 447 467
29 26 32
2.4 2.1 2.4
438 457 451
24 29 27
2.4 2.5 2.3
418 456 449
18 29 27
2.4 2.3 2.4
Supplement to basal ration
5.0% A + 5.0% W 7.5% A + 7.5% W 7.5% A+10.0% W Av. Alfalfa Meal Av. Dried Whey Av. Alfalfa+Whey
* Average of two replicates of eight males and eight females each.
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ground limestone and 0.023 magnesium carbonate, the percentage mineral content of the basal ration calculated as follows: calcium 1.42, phosphorus 0.99, potassium 0.3, sodium chloride 0.74 and magnesium 0.049. Other mineral additions per pound of ration were 4.3 mg. Zn as ZnCl2, 0.07 mg. B as H 3 B, 0.05 mg. Co as CoS0 4 .7H 2 0, 0.5 mg. I as KI, 84 mg. Mn as Mn SO4.H2O and trace mineral salt and 11 mg. Fe and 1.1 mg. Cu as trace mineral salt. Adequacy of the diet in certain known vitamins, amino acids and essential minerals was checked by doubling the intake of riboflavin, thiamine, and calcium pantothenate and the trace minerals, increasing added magnesium to 200 mg. per pound, and adding singly or combined 0.1 percent tryptophane, 0.2 percent arginine, 0.1 percent methionine, 0.5 percent glycine and the single addition of 5% "vitamin-free" casein. None of these additional supplements increased growth. Chicks were maintained in batteries in a room with thermostatically controlled heat (±2°F.) and good air circulation. Room temperature in cold weather was held at 90°F. for the first week, lowered to 85°F. for a week and held thereafter at 75°F. Chicks were hatched on a Friday and fed the unsupplemented ration until the following Monday. Chicks were then allotted to duplicate pens on the basis of sex and weight using eight males and eight females per pen. Thus, 32 chicks were started on each dietary treatment or lot. Unless otherwise stated, deaths were never greater than two chicks per lot. Feed and water were available ad libitum for the four week experimental period. Chicks were individually weighed at the start and at the end of the 2nd, 3rd and 4th weeks. Final average weights for a treatment were made by equalizing male and female weights followed by equalizing pen or lot weights.
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UNIDENTIFIED GROWTH FACTORS TABLE 2.—Average responses in six replicated trials (192 chicks per supplement) from alfalfa •\-whey and distillers solubles
Supplement to basal ration 5% Alfalfa M e a l + 5 % Dried Whey (AW) 10% Distillers Dried Solubles (DDS) 10%AW+10%DDS
%gain over Feed/Wt. controls gain (4-wks.)* 16
2.4
19 29
2.3 2.3
of different factors in these two supplements. Fish solubles (FS) was studied over a range of 3 to 7 percent of the ration. Three percent FS gave an 8 percent increase in weight over the basal. This was not appreciably improved by the higher levels of FS. Table 2 shows typical responses obtained with AW and DDS singly and in combination. Six replicated trials gave substantially the same results and point to the presence in DDS of a factor distinct from that in alfalfa or whey. Table 3 summarizes results secured from AW, LR and DDS alone and in combination. These data indicate that LR contains a growth factor not common to AW or DDS. Ash of DDS prepared at 700°C. and added at a 10% equivalence produced a slight response (Table 4) but the difference was no greater than that secured TABLE 3.—Chick responses to the feeding of various carriers of unidentified factors. (32 chicks per supplement) Supplement to basal ration
Av. 4-wk. wt. (gms.)
None
232
10% DDS 5% Liver Residue (LR) 10% AW 10% AW+10% DDS 5% L R + 1 0 % DDS 5%LR+10%AW 5% LR +10% AW+10% DDS
287 360 287 357 403 420 455
%
gain Feed/Wt. over gain controls — 3.7 24 55 24 54 74 81 96
3.2 2.6 3.3 2.8 2.6 2.4 2.4
Supplement to basal ration
Av. 4-wk. wt. (gms.)
None 10% DDS Ash of 10% DDS
Feed/Wt. gain
331 369 (11%)* 340 (3%)*
2.5 2.4 2.S
* Over controls.
from duplicate lots fed the basal ration in the same trial. Table 5 summarizes the results of a complete four-member factorial experiment aimed at determining the relationship between the factors of FS, AW, LR and DDS. This trial again points to the existence of three separate entities. FS appears to contain the same factor as LR but lacks both DDS and AW activity. Furthermore, LR appears to furnish some AW factor while DDS contains some LR factor. It will be noted that the combined fourmember supplement, AW, FS, LR and DDS failed to equal the growth obtained TABLE 5.—Average chick responses in a four-member factorial study of alfalfa+whey, fish solubles, liver residue, and distillers dried solubles. (32 chicks per supplement)
Supplements to basal ration
Av. 4-wk. wt. (gms.)
%
gain F e e d / W t . gain over controls
None
390
—
2.6
10% D D S 10% AW 3 % Fish Solubles (FS) 5%LR
495 497 416 508
27 27 7 30
2.3 2.5 2.4 2.2
10% 10% 10% 10% 10% 3%
D D S + 1 0 % AW D D S + 3 % FS D D S + 5 % LR A W + 3 % FS A W + 5 % LR F S + 5 % LR
540 542 542 505 533 532
38 39 39 29 37 36
2.3 2.2 2.1 2.4 2.2 2.1
10% 10% 10% 10%
D D S + 1 0 % A W + 3 % FS D D S + 3 % F S + 5 % LR D D S + 1 0 % A W + 5 % LR A W + 3 % F S + 5 % LR
531 563 569 543
36 44 46 39
2.5 2.2 2.2 2.3
530
36
2.4
563
44
2.1
10% A W + 3 % F S + 5 % L R + 10% D D S 10% A W + 3 % F S + 5 % L R + 1 0 % D D S + 1 0 % AF*
* Stabilized prime white grease.
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* Average weight of controls 326 grams with feed efficiency of 2.6.
TABLE 4.—Average chick responses secured from two replicated trials with distillers dried solubles and the ash of distillers dried solubles. (64 chicks per supplement)
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R. A. RASMUSSEN, P. W. LUTHY, J. M. VAN LANEN AND C. S. BORUFF
ably over those secured from the rations not adjusted for caloric content. The greatest difference between the nonadjusted and the adjusted ration was 3.6 percent. The most notable favorable effect was on feed efficiency which improved both on an equicaloric and a calorie-toprotein basis. Except for LR the protein of the supplements appears to be of too low quality and supplementary value to support adjustment of calories to supplement protein at the 41 Calories of productive energy per pound for each percent protein. The other supplements resulted in decreased gains for each ration. This ration plus LR produced similar responses at the three caloric levels, but the better protein quality of LR as compared to the other supplements did not significantly increase chick gains. DISCUSSION The results reported in Table 1 indicate that alfalfa and whey contain the same factor. This is based upon the observation that alfalfa and whey together produced no greater growth than whey alone
TABLE 6.—Average chick responses in a three-member factorial employing equicaloric and 41 Calories of productive energy per pound per percent protein. Two lots with no caloric adjustment are included for comparison Av. 4-week weights (gms.)
None 10% DDS 10% AW 5%LR 10% D D S + 1 0 % AW 10% D D S + 5% LR 10% AW + 5% LR 10% D D S + 1 0 % A W + 5 % LR
No fat added
Cal. P.E. equal
41 Cal. P.E. per lb. per % prot.
(1)
(2)
(3)
(1)
(2)
(3)
386
386
386
2.5
2.5
2.5
ill
Supplements to basal ration
495 492 517
464 485 520
2.2 (2.5)* (2.2)*
2.1 2.3 2.0
2.0 2.0 1.8
515 (542)* (533)*
522 556 542
480 526 480
2.2 (2.1)* (2.2)*
2.0 2.0 1.9
1.9 1.7 1.8
558
578
331t
2.1
1.8
—
* Parenthesis values from Table 5 included for comparison. 1 4 1 % of the chicks failed to survive the experimental period.
Feed/Weight gain
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with AW, LR and DDS. The productive energy per pound of ration was 936 Calories for the three-member supplement, 912 Calories for the four-member supplement and 1,077 Calories for the basal ration. Thus feed utilization and growth rates were improved with these supplements despite the decline in caloric content (Table 5). This suggested that equicaloric rations might allow these supplements to be more effective. In Table 6 are reported the results secured from two three-member factorial experiments in which: 1) the caloric content of the rations was held constant at the level of the basal ration (1,077 Calories of productive energy per pound) by addition of animal fat at the expense of Cerelose and 2) the Calories of productive energy per pound of ration were related to the protein in the ratio of 41 Calories for each percent protein by multiplying the total protein (basal+supplements) by 41 and adding animal fat at the expense of Cerelose to give the desired Calories. Weight gains with the equicaloric rations containing AW or LR supplements (Table 6) were improved but not appreci-
UNIDENTIFIED GROWTH FACTORS
The existence in distillers dried solubles of a second growth factor distinct from that in alfalfa and whey is indicated in Table 2. This finding is in agreement with Norris (1954) and Scott (1955); but not with the reports of Couch et al. (1952) and Reid et al. (1956). The distillers solubles factor is not "vitamin Bi 3 " as the activity also is present in distillers solubles from which "vitamin Bi 3 " has been extracted. The existence of a third factor in liver residue (Wilson) is indicated in Table 3. This factor produced additional growth over all the single supplements and also over the combination of alfalfa-whey and distillers dried solubles. The presence of a growth factor in liver is in agreement with the findings of Fisher et al. (1954), Norris (1954), Combs et al. (1954) and Rasmussen el al. (1954). In addition to the above supplements, chicks on the basal ration described respond to "vitamin Bi3" concentrate, distillers dried solubles from which "vitamin Bi 3 " has been extracted, BY-100, penicillin mycelium, corn steep liquor and animal peptone. No response was obtained with molasses distillers solubles, Vigofac or the ash of distillers solubles.
The ash of liver residue and alfalfa-whey were not investigated. Gelatin and blood fibrin which contain active ash (Benton et al., 1955b) are present in our basal ration. The four-member factorial study reported in Table 5 indicates that the fish solubles factor is contained in liver residue. This agrees with the finding of similar factors in a liver fraction and condensed fish solubles by Combs et al. (1954) and in whole liver powder and fish meal by Tamimie (1955). The presence of lesser amounts of alfalfa-whey factor in liver residue and distillers solubles, and a relatively small amount of either distillers solubles factor or liver factor in alfalfa-whey can account for the lower growth response secured from fish solubles in relation to the other three individual supplements (Table 5). Results with two-member additions indicate that the alfalfa-whey supplement contained some liver residue factor but no appreciable amount of the distillers solubles factor. This is indicated by the somewhat lower maximum response from the alfalfa-whey plus fish solubles. The three-member supplements (Table 5) ( D D S + F S + L R a n d D D S + A W + L R ) produced definitely increased growth over the two-member additions whereas DDS + A W + F S and A W + F S + L R and the four-member supplements failed to produce expected gains. Lower caloric content in the multiple-supplemented rations was indicated and the addition of 10% fat to the four-member supplement supported this conclusion. When productive energy was not adjusted (Table 6), energy again seemed to limit growth with the two-member and three-member supplements. Better gains with equicaloric rations over those not adjusted for productive energy is most likely a further expression of unidentified factors. Increase in feed efficiency appears
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and from other trials in which half-maximum response levels of alfalfa and whey gave growth equivalent to the maximum from whey. Failure to secure an alfalfa meal response equal to whey may be due to the alfalfa inhibitor (Lepkovsky et al., 1950). The finding of a single factor in alfalfa and whey does not agree with the observations of Fisher et al. (1954) or Combs et al. (1954) and may be due to differences in the basal rations. Combs et al. (1954) indicate the presence of more than one factor in alfalfa meal. The blood meal and other natural materials used in our basal diet may contain such a second factor. Our results, however, demonstrate the presence of "whey factor" in alfalfa meal.
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52
R. A. RASMUSSEN, P. W. LUTHY, J,. M. VAN LANEN AND C. S. BORUFF
The relationship of the factors herein reported to the grass juice factor (Norris, 1954) can only be speculative; however, as fish solubles and distillers solubles are common to both studies it is possible that the grass juice factor is similar to this study's alfalfa-whey factor. The relationship of the brewers yeast and molasses distillers solubles factors of Combs et al. (1954) to the present factors await further study. With our basal ration, a chick growth response is not obtained with molasses distillers solubles. This and the other factors mentioned
which give no response may be supplied by the basal diet or carried over from the maternal diet. With gelatin and blood meal in the basal diet it is unlikely that a response to the gelatin factor (Benton et al., 1955) would be obtained. The three unidentified factors reported here have also been demonstrated by Fisher et al. (1954) and Scott (1955). SUMMARY
An inexpensive basal diet considered to be adequate in known nutritional essentials has been devised and used in experimental chick studies of unidentified factors required by the non-depleted chick. It produces good growth and permits the separation of unidentified factors as indicated by increased growth responses with various combinations of unidentified factor carriers fed at maximum response levels. Evidence which suggests that three different unidentified factors are essential for rapid growth of chicks has been found, viz.: 1) A factor present in alfalfa meal and dried whey. 2) A factor present in Wilson's liver residue and fish solubles. 3) A factor present in distillers dried solubles. With the exception of the fish solubles each of these unidentified factor carriers appears to contain lesser amounts of the other factors. Fish solubles appears to contain only the liver residue factor. When the productive energy was maintained at the level of the basal ration (1,077 Calories per pound) the unidentified factors in the various supplements were more fully expressed. ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Virgil L. Boone and William H. McDaniels in carrying out
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to be the major effect of additional calories if a ration contains 1,000 Calories or more of P.E. per pound and more than adequate amount of other required nutrients. This increased feed efficiency may be accompanied by a small increase in weight gain. This is true in the case of liver residue (Table 6) and is indicated in the studies of Combs and Romoser (1955), wherein differences of gains of 1.6 percent are reported between 1,087 and 1,240 Calories P.E. per pound of ration while feed efficiencies were improved from 1.72 to 1.60 pounds of feed per pound gain. The energy value of a ration is a primary factor in regulating consumption of feed (Hill and Dansky, 1954; and Mellen et al., 1954). Thus decreased growth was encountered when the rations (Table 6) were equalized in their ratio of productive energy to protein, because the high calorie content of the rations decreased intake of some nutrient(s) below that required for maximum growth. For three-member supplements, over 29% of fat was required to maintain the productive energy per pound at 41 Calories per percent protein. On these diets most deaths occurred during the first week. Whether fat per se, a dietary imbalance or some other factor caused the deaths could not be ascertained.
UNIDENTIFIED GROWTH FACTORS
this study. They are indebted also to Mr. H. C. Schaefer of the Ralston-Purina Co. for the fish solubles used. The liver residue was obtained from Wilson and Company, Chicago, Illinois, and is the residue of minced whole liver extracted with acidulated water and combined with the heat-coagulated residue of the extract. REFERENCES
Further studies on the alfalfa factor and its relation to the liver and whey factors. / . Nutrition, 52:13-24. Hill, F. W., and L. M. Dansky, 1954. Studies of the energy requirements of chickens. 1. The effect of dietary energy level on growth and feed consumption. Poultry Sci. 33: 112-119. Kodras, R., W. I. Cooney and J. S. Butts, 1951a. Chick growth depressing factor in sun-cured and dehydrated alfalfa meals. Poultry Sci. 30: 280-291. Kodras, R., W. I. Cooney and J. S. Butts, 1951b. Effect of alfalfa meal, alfalfa leaves, alfalfa stems and fresh alfalfa on chick growth. Poultry Sci. 30: 768-787. Kohler, G. O., and W. R. Graham, Jr., 1951. A chick growth factor found in leafy green vegetation. Poultry Sci. 30:484-491. Lepkovsky, S., W. Shaeleff, D. Peterson and R. Perry, 1950. Alfalfa inhibitor in chick rations. Poultry Sci. 29:208-213. March, B., J. Biely and S. P. Touchburn, 1955. Studies on an unidentified chick growth factor in dehydrated green feed. Poultry Sci. 34: 968-972. Mellen, W. J., F. W. Hill and H. H. Dukes, 1954. Studies of the energy requirements of chickens. 2. Effect of dietary energy level on the basal metabolism of growing chickens. Poultry Sci. 33: 791-798. Menge, H., G. F. Combs, Peng-Tung Hsu and M. S. Shorb, 1952. Unidentified growth factors required by chicks and poults. 1. Studies with chicks using purified diets. Poultry Sci. 31: 237-247. Norris, L. C , 1954. Unidentified chick growth factors in distillers dried solubles. Proc. 8th Distillers Feed Research Conference, pp 20-28. Norris, L. C , 1955. Recent progress on unidentified growth factors in distillers dried solubles and other feed supplements. Proc. 10th Distillers Feed Research Conference, pp 39-42. Petersen, C. F., A. C. Wiese and A. R. Pappenhagen, 1955. Unidentified chick growth factors. 1. Purified assay diet and crude supplement response. Poultry Sci. 34:673-678. Rasmussen, R. A., P. W. Luthy, J. M. Van Lanen and C. S. Boruff, 1954. The dual nature of the unidentified chick growth promoting activity of distillers dried solubles. Proc. 9th Distillers Feed Research Conference, pp 29-37. Reid, B. L., R. L. Svacha, A. A. Kurnick, F. M. Salama and J. R. Couch, 1956. Unidentified growth factors and thioctic acid in chick and poult nutrition. Distillers Feed Conference 11: 68-79.
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Arscott, G. H., and G. F. Combs, 1955. Unidentified growth factors required by chicks and poults. 4. Experimental variables which influence the dietary requirements of chicks for these factors. Poultry Sci. 34: 843-849. Benton, D. A., H. E. Spivey, A. E. Harper and C. A. Elvehjem, 1955a. Factors affecting the growth of chicks on amino acid diets. Arch. Biochem. Biophys. 57: 262-263. Benton, D. A., H. E. Spivey, A. E. Harper and C. A. Elvehjem, 1955b. Gelatin as a source of factors needed by chicks on amino acid diets. Abstr. of papers presented 128th Meeting of Am. Chem. Soc, Minneapolis, Minn., p 10c. Carlson, C. W., R. F. Miller, H. T. Peeler, L. C. Norris and G. F. Heuser, 1949. The complex nature of the animal protein factor. Poultry Sci. 28:750-752. Combs, G. F., G. B. Sweet, H. J. Jones, G. L. Romoser and R. W. Bishop, 1954. Multiplicity of unidentified growth factors required by chicks and poults. Poultry Sci. 33:1050. Combs, G. E., 1955. Recent advances in poultry nutrition. Annual Meeting of Assoc, of Amer. Feed Control Officials, Wash., D. C. (in Feedstuffs 27, No. 46: 39-40,45-50). Combs, G. F., and G. L. Romoser, 1955. A new approach to poultry feed formulation. Feed Age, 5, No. 3:50-58. Couch, J. R., J. R. Reed, Jr., B. L. Reid and J. W. Diekert, 1952. Distillers solubles and other sources of unidentified factors for promoting the growth of chicks and poults. Proc. 7th Distillers Feed Research Conference, pp. 59—71. Couch, J. R., B. L. Reid, A. A. Camp, W. N. Dannenburg and E. E. Rozacky, 1955. Distillers dried solubles in the diets of chicks. Proc. 10th Distillers Feed Research Conference pp. 47-57. Edwards, H. M., R. Dam, L. C. Norris and G. F. Heuser, 1953. Probable identity of unidentified chick growth factors in fish solubles and penicillin mycelium residue. Poultry Sci. 32: 551-554. Fisher, H., H. M. Scott and R. G. Hansen, 1954.
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Scott, H. M., W. D. Morrison and P. Griminger, 1955. Studies on unknown growth factors in distillers dried solubles. Poultry Sci. 34: 14461447. Stokstad, E. L. R., and T. H. Jukes, 1950. Further observations on the "animal protein factor." Proc. Soc. Exptl. Biol. Med. 73:523-528. Sunde, M. L., W. W. Cravens, C. A. Elvehjem and J. G. Halpin, 1950. An unidentified factor re-
quired by chicks fed practical rations. Poultry Sci. 29: 204-207. Tamimie, H. S., 1955. The response of chicks to an unidentified growth factor in fish products. Poultry Sci. 34:1224. Titus, H. W., 1955. The Scientific Feeding of Chickens. Energy values of feedstuffs for poultry. Revision of 2nd Ed. with Addendum. The Interstate, Danville, Illinois, pp 258-261.
2. THE IMMUNE RESPONSE OF CHICKENS VACCINATED WITH Bl NEWCASTLE DISEASE VIRUS ADMINISTERED THROUGH THE DRINKING WATER* R. W. WINTERFIELD AND E. H. SEADALE Department of Veterinary Science, University of Massachusetts, Amherst, Mass. (Received for publication July 9, 1956)
T
HIS investigation was undertaken to further study and to determine the efficacy of mass vaccination of chickens for Newcastle disease (ND) via the drinking water (Luginbuhl et al., 1954; Winterfield and Seadale, 1955; Luginbuhl et al., 1955). Factors having a possible influence on immunity induced from this route of administration were studied, and the results are presented. MATERIALS AND METHODS
Bl strain Newcastle disease virus (NDV) (Hitchner and Johnson, 1948) vaccines, designated A, B, and C, used in all trials, were obtained from three sources. Vaccine A was a lyophilized product of commercial origin. It was supplied in 500dose vials accompanied by 15 ml. diluent per vial in separate, plastic containers. It was suggested that chicks 3 to 21 days of age be vaccinated after being deprived of water for 4 hours. The dried virus was reconstituted with the diluent and im* Contribution No. 1042 of the Massachusetts Agricultural Experiment Station.
mediately added to the drinking water in the ratio of 1:315.3. Vaccine B, also of commercial origin, was a glycerinated virus preparation supplied in 60-ml., 1,000dose quantities per bottle with an accompanying package of dried, nutrient stabilizer. One ml. of the vaccine was added to 315.3 ml. of water after a premixture had been made with 30 ml. of water and .41 gram of stabilizing material. Vaccine C represented Bl—NDV lyophilized in the authors' laboratory in 3-ml. quantities per vial from a 1:1 mixture of 10 percent skim milk and infected amnioallantoic fluid. It was reconstituted and diluted in the drinking water in the manner described with vaccine A. A stabilizer composed of two-thirds gelatin and one-third dried skim milk was added in the ratio of 1.3 grams to 315.3 ml. of drinking water. Previous studies (Winterfield and Seadale, 1956) demonstrated the ability of a stabilizer to maintain higher vaccine titers in water under simulated conditions of field administration. Single distilled water was used in all trials. Titrations of vaccines and vaccine-
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Newcastle Disease Immunization Studies
T
* Present address: Peter Hand Foundation, Chicago 22, Illinois.
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tors. Fisher et al. (1954) postulate three distinct chick growth factors present in alfalfa meal, dried whey, and defatted whole liver powder, respectively. A further report by Scott (1955) from the same laboratory adds a fourth factor found in distillers dried solubles. Rasmussen et al. (1954) reported the presence of two unidentified chick growth factors in distillers dried solubles. One factor is "vitamin Bi3" and the other produces responses of about the same magnitude as those secured from liver residue. Combs et al. (1954) found the chick to require three unidentified growth factors—one found in condensed fish solubles and liver fraction NF 2 (Armour and Company); the second in dried whey products, dried molasses distillers solubles, and dried brewers yeast; and the third in dehydrated alfalfa meal and dried brewers yeast. Peterson et al. (1955) reported a factor common to herring fish meal, sardine fish solubles, 50% dried lactose whey product, BioPar "C," and defatted liver residue. All of these items produced equal but not additive growth responses when added to their basal purified ration. Arscott and Combs (1955) noted that condensed fish solubles contains two unidentified factors, one of which is present in a liver fraction and the other in dehydrated alfalfa meal. March et al. (1955) elucidated two unidentified chick growth factors in dehydrated cereal grass and herring meal. The response to either was dependent on the presence of the other in the ration.
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HE presence of unidentified nutritional factors in various ingredients fed to chicks has been well established. Much of the literature on this subject has been reviewed by Menge et al. (1952). Considerable evidence has accumulated pointing to a multiplicity of unidentified factors required by the chick. Carlson et al. (1949) postulated the presence of two distinct factors, one in fish meal and the other in dried brewers yeast. Stokstad and Jukes (1950) reported that at least part of the chick response to Streptomyces aureofaciens materials was due to chlortetracycline. Kohler and Graham (1951) concluded that the unidentified forage crops juice factor is different from vitamin B13, whey factor, or antibiotic effect. Couch et al. (1952) found one unidentified chick factor in distillers dried solubles, dried whey, and whey product and another in fish meal. Edwards et al. (1953) reported that fish solubles and penicillin mycelium residue contain the same unidentified chick growth factor(s). Menge et al. (1952) found that two distinct unidentified factors are required by the chick for rapid growth. One of these is present in liver fraction "L," liver fraction BioPar " C , " and dried brewers yeast, and the other in dried whey or dried whey products. Norris (1954) reported that fish solubles, distillers dried solubles, and grass juice each contain distinct unidentified growth fac-
UNIDENTIFIED GROWTH FACTORS
EXPERIMENTAL PROCEDURE
New Hampshire X Indian River or Columbian crossbred chicks were used in all experiments. These chicks were progeny of dams kept in individual-pen laying batteries with raised wire floors. During the period in which eggs were collected for incubation the hens were artificially inseminated twice weekly with fresh, pooled semen from males kept in individual-pen batteries with raised wire floors. Little was gained by depletion of the hens through feeding an all vegetableprotein diet. Both controls and supplemented chicks from depleted hens were correspondingly smaller at four weeks than chicks from hens fed rations con-
taining animal proteins. Therefore, the breeder diet for these studies was fortified with unidentified factor carriers. The breeder diet had the following percentage composition: 65.75 yellow corn, 14 Hi-Pro-Con (50% protein soybean oil meal), 3 meat and bone scraps, 2 fish meal, 2 dried whey, 3 distillers dried solubles, 3 alfalfa meal, 0.2 viadex (4,000A750D), 3 dicalcium phosphate, 3.5 limestone, 0.5 trace mineral salt, 0.0066 manganese sulfate, 0.025 diphenyl-pphenylenediamine, 0.025 riboflavin supplement (8,000 mcg./gm.), 0.00147 niacin, and 0.00088 calcium pantothenate. Oyster shell was available ad libitum. The basal chick diet had the following percentage composition: 56.18 Cerelose, 0.25 corn oil, 17.5 blood meal, 14 Hi-ProCon, 3.5 gelatin (Keystone No. 11 edible), 2 Stamino type A (A. E. Staley Company containing 10% isoleucine), 0.3 methionine, 1.1 vitamin mixture, 0.5% trace mineralized salt (Hardy formula #2) and 4.67 minerals. This basal contained 27.8 percent protein and 1,077 Calories of productive energy (P.E.) per pound (Titus, 1955). By calculation all essential amino acids not specifically tested and found adequate were present in excess of NRC requirements adjusted for a ration of 27.8% protein. The vitamin mixture furnished per kilogram of ration the following: 6,040 I.U. vitamin A, 1,130 I.U. vitamin D3, 2 gm. choline chloride, 0.2 mg. vitamin Bi2, 20 mg. thiamine, 7 mg. riboflavin, 20 mg. calcium pantothenate, 32 mg. niacin, 7 mg. pyridoxin, 0.2 mg. biotin, 1.4 mg. folic acid, 1 mg. Klotogen F (sodium bisulfite salt of Menadione), 2 mg. paminobenzoic acid, 100 mg. inositol, 20 mg. alpha-tocopherol acetate, 100 mg. ascorbic acid, and 250 mg. diphenyl-pphenylenediamine. With 4% dicalcium phosphate, 0.6%
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Norris (1955) and Couch et al. (1955) reported that at least part of the unidentified factor response secured from distillers dried solubles and other unidentified factor supplements is obtained from the ash of these supplements. Benton et al. (1955a,b) indicated that half of the growth activity of gelatin was due to an ash component. Combs (1955) reported that, under his conditions, distillers solubles ash failed to produce a response but that a combination of ash from fish solubles, dried whey and alfalfa meal promoted chick growth slightly. Scott el al. (1955) secured a significant chick growth response to distillers solubles but not to distillers solubles ash. Fisher et al. (1954) found an unidentified factor in alfalfa not associated with alfalfa ash. This paper presents results of studies conducted since June of 1953 on the unidentified factors required for chick growth employing a relatively inexpensive semipurified diet which produces good growth. At least three different unidentified factors are evident with this ration.
47
48
R. A. RASMUSSEN, P. W. LUTHY, J. M. VAN LANEN AND C. S. BORUFF
Additions to the basal ration were made at the expense of Cerelose. RESULTS
Growth responses with graded levels of alfalfa and whey are shown in Table 1. These results reveal no additive effect of alfalfa (A) and dried whey (W). The alfalfa meal (17 percent protein, artificially dried) did not equal dried whey at equivalent levels nor did it sharply depress growth at the higher levels as observed by others, (Lepkovsky et al., 1950; and Kodras et al, 1951a, b). However, continued trials with these two supplements singly and in combination suggests that the same factor is supplied by both but that alfalfa reaches a lower maximum response because of its "inhibitor" content. In some trials 10 percent alfalfa gave a definite growth depression. The very similar responses from alfalfa and whey and from whey alone indicate a single factor. In subsequent experiments, 5 percent alfalfa and 5 percent whey which gave near maximum response were employed as a single factor source (AW). Other investigators (Fisher el al., 1954; and Combs et al., 1954) observed a growth differential between alfalfa meal and dried whey pointing to the existence TABLE 1.—Growth responses of chicks to alfalfa and whey supplementation Av. 4-wk. wt.* (gms.)
% gain over controls
Feed/Wt. gain
None
354
-
2.6
5 . 0 % Alfalfa Meal (A) 7.5% A • 10.0% A
422 411 422
19 16 19
2.5 2.4 2.3
5 . 0 % Dried Whey (W) 7.5% W 10.0% W
455 447 467
29 26 32
2.4 2.1 2.4
438 457 451
24 29 27
2.4 2.5 2.3
418 456 449
18 29 27
2.4 2.3 2.4
Supplement to basal ration
5.0% A + 5.0% W 7.5% A + 7.5% W 7.5% A+10.0% W Av. Alfalfa Meal Av. Dried Whey Av. Alfalfa+Whey
* Average of two replicates of eight males and eight females each.
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ground limestone and 0.023 magnesium carbonate, the percentage mineral content of the basal ration calculated as follows: calcium 1.42, phosphorus 0.99, potassium 0.3, sodium chloride 0.74 and magnesium 0.049. Other mineral additions per pound of ration were 4.3 mg. Zn as ZnCl2, 0.07 mg. B as H 3 B, 0.05 mg. Co as CoS0 4 .7H 2 0, 0.5 mg. I as KI, 84 mg. Mn as Mn SO4.H2O and trace mineral salt and 11 mg. Fe and 1.1 mg. Cu as trace mineral salt. Adequacy of the diet in certain known vitamins, amino acids and essential minerals was checked by doubling the intake of riboflavin, thiamine, and calcium pantothenate and the trace minerals, increasing added magnesium to 200 mg. per pound, and adding singly or combined 0.1 percent tryptophane, 0.2 percent arginine, 0.1 percent methionine, 0.5 percent glycine and the single addition of 5% "vitamin-free" casein. None of these additional supplements increased growth. Chicks were maintained in batteries in a room with thermostatically controlled heat (±2°F.) and good air circulation. Room temperature in cold weather was held at 90°F. for the first week, lowered to 85°F. for a week and held thereafter at 75°F. Chicks were hatched on a Friday and fed the unsupplemented ration until the following Monday. Chicks were then allotted to duplicate pens on the basis of sex and weight using eight males and eight females per pen. Thus, 32 chicks were started on each dietary treatment or lot. Unless otherwise stated, deaths were never greater than two chicks per lot. Feed and water were available ad libitum for the four week experimental period. Chicks were individually weighed at the start and at the end of the 2nd, 3rd and 4th weeks. Final average weights for a treatment were made by equalizing male and female weights followed by equalizing pen or lot weights.
49
UNIDENTIFIED GROWTH FACTORS TABLE 2.—Average responses in six replicated trials (192 chicks per supplement) from alfalfa •\-whey and distillers solubles
Supplement to basal ration 5% Alfalfa M e a l + 5 % Dried Whey (AW) 10% Distillers Dried Solubles (DDS) 10%AW+10%DDS
%gain over Feed/Wt. controls gain (4-wks.)* 16
2.4
19 29
2.3 2.3
of different factors in these two supplements. Fish solubles (FS) was studied over a range of 3 to 7 percent of the ration. Three percent FS gave an 8 percent increase in weight over the basal. This was not appreciably improved by the higher levels of FS. Table 2 shows typical responses obtained with AW and DDS singly and in combination. Six replicated trials gave substantially the same results and point to the presence in DDS of a factor distinct from that in alfalfa or whey. Table 3 summarizes results secured from AW, LR and DDS alone and in combination. These data indicate that LR contains a growth factor not common to AW or DDS. Ash of DDS prepared at 700°C. and added at a 10% equivalence produced a slight response (Table 4) but the difference was no greater than that secured TABLE 3.—Chick responses to the feeding of various carriers of unidentified factors. (32 chicks per supplement) Supplement to basal ration
Av. 4-wk. wt. (gms.)
None
232
10% DDS 5% Liver Residue (LR) 10% AW 10% AW+10% DDS 5% L R + 1 0 % DDS 5%LR+10%AW 5% LR +10% AW+10% DDS
287 360 287 357 403 420 455
%
gain Feed/Wt. over gain controls — 3.7 24 55 24 54 74 81 96
3.2 2.6 3.3 2.8 2.6 2.4 2.4
Supplement to basal ration
Av. 4-wk. wt. (gms.)
None 10% DDS Ash of 10% DDS
Feed/Wt. gain
331 369 (11%)* 340 (3%)*
2.5 2.4 2.S
* Over controls.
from duplicate lots fed the basal ration in the same trial. Table 5 summarizes the results of a complete four-member factorial experiment aimed at determining the relationship between the factors of FS, AW, LR and DDS. This trial again points to the existence of three separate entities. FS appears to contain the same factor as LR but lacks both DDS and AW activity. Furthermore, LR appears to furnish some AW factor while DDS contains some LR factor. It will be noted that the combined fourmember supplement, AW, FS, LR and DDS failed to equal the growth obtained TABLE 5.—Average chick responses in a four-member factorial study of alfalfa+whey, fish solubles, liver residue, and distillers dried solubles. (32 chicks per supplement)
Supplements to basal ration
Av. 4-wk. wt. (gms.)
%
gain F e e d / W t . gain over controls
None
390
—
2.6
10% D D S 10% AW 3 % Fish Solubles (FS) 5%LR
495 497 416 508
27 27 7 30
2.3 2.5 2.4 2.2
10% 10% 10% 10% 10% 3%
D D S + 1 0 % AW D D S + 3 % FS D D S + 5 % LR A W + 3 % FS A W + 5 % LR F S + 5 % LR
540 542 542 505 533 532
38 39 39 29 37 36
2.3 2.2 2.1 2.4 2.2 2.1
10% 10% 10% 10%
D D S + 1 0 % A W + 3 % FS D D S + 3 % F S + 5 % LR D D S + 1 0 % A W + 5 % LR A W + 3 % F S + 5 % LR
531 563 569 543
36 44 46 39
2.5 2.2 2.2 2.3
530
36
2.4
563
44
2.1
10% A W + 3 % F S + 5 % L R + 10% D D S 10% A W + 3 % F S + 5 % L R + 1 0 % D D S + 1 0 % AF*
* Stabilized prime white grease.
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* Average weight of controls 326 grams with feed efficiency of 2.6.
TABLE 4.—Average chick responses secured from two replicated trials with distillers dried solubles and the ash of distillers dried solubles. (64 chicks per supplement)
50
R. A. RASMUSSEN, P. W. LUTHY, J. M. VAN LANEN AND C. S. BORUFF
ably over those secured from the rations not adjusted for caloric content. The greatest difference between the nonadjusted and the adjusted ration was 3.6 percent. The most notable favorable effect was on feed efficiency which improved both on an equicaloric and a calorie-toprotein basis. Except for LR the protein of the supplements appears to be of too low quality and supplementary value to support adjustment of calories to supplement protein at the 41 Calories of productive energy per pound for each percent protein. The other supplements resulted in decreased gains for each ration. This ration plus LR produced similar responses at the three caloric levels, but the better protein quality of LR as compared to the other supplements did not significantly increase chick gains. DISCUSSION The results reported in Table 1 indicate that alfalfa and whey contain the same factor. This is based upon the observation that alfalfa and whey together produced no greater growth than whey alone
TABLE 6.—Average chick responses in a three-member factorial employing equicaloric and 41 Calories of productive energy per pound per percent protein. Two lots with no caloric adjustment are included for comparison Av. 4-week weights (gms.)
None 10% DDS 10% AW 5%LR 10% D D S + 1 0 % AW 10% D D S + 5% LR 10% AW + 5% LR 10% D D S + 1 0 % A W + 5 % LR
No fat added
Cal. P.E. equal
41 Cal. P.E. per lb. per % prot.
(1)
(2)
(3)
(1)
(2)
(3)
386
386
386
2.5
2.5
2.5
ill
Supplements to basal ration
495 492 517
464 485 520
2.2 (2.5)* (2.2)*
2.1 2.3 2.0
2.0 2.0 1.8
515 (542)* (533)*
522 556 542
480 526 480
2.2 (2.1)* (2.2)*
2.0 2.0 1.9
1.9 1.7 1.8
558
578
331t
2.1
1.8
—
* Parenthesis values from Table 5 included for comparison. 1 4 1 % of the chicks failed to survive the experimental period.
Feed/Weight gain
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with AW, LR and DDS. The productive energy per pound of ration was 936 Calories for the three-member supplement, 912 Calories for the four-member supplement and 1,077 Calories for the basal ration. Thus feed utilization and growth rates were improved with these supplements despite the decline in caloric content (Table 5). This suggested that equicaloric rations might allow these supplements to be more effective. In Table 6 are reported the results secured from two three-member factorial experiments in which: 1) the caloric content of the rations was held constant at the level of the basal ration (1,077 Calories of productive energy per pound) by addition of animal fat at the expense of Cerelose and 2) the Calories of productive energy per pound of ration were related to the protein in the ratio of 41 Calories for each percent protein by multiplying the total protein (basal+supplements) by 41 and adding animal fat at the expense of Cerelose to give the desired Calories. Weight gains with the equicaloric rations containing AW or LR supplements (Table 6) were improved but not appreci-
UNIDENTIFIED GROWTH FACTORS
The existence in distillers dried solubles of a second growth factor distinct from that in alfalfa and whey is indicated in Table 2. This finding is in agreement with Norris (1954) and Scott (1955); but not with the reports of Couch et al. (1952) and Reid et al. (1956). The distillers solubles factor is not "vitamin Bi 3 " as the activity also is present in distillers solubles from which "vitamin Bi 3 " has been extracted. The existence of a third factor in liver residue (Wilson) is indicated in Table 3. This factor produced additional growth over all the single supplements and also over the combination of alfalfa-whey and distillers dried solubles. The presence of a growth factor in liver is in agreement with the findings of Fisher et al. (1954), Norris (1954), Combs et al. (1954) and Rasmussen el al. (1954). In addition to the above supplements, chicks on the basal ration described respond to "vitamin Bi3" concentrate, distillers dried solubles from which "vitamin Bi 3 " has been extracted, BY-100, penicillin mycelium, corn steep liquor and animal peptone. No response was obtained with molasses distillers solubles, Vigofac or the ash of distillers solubles.
The ash of liver residue and alfalfa-whey were not investigated. Gelatin and blood fibrin which contain active ash (Benton et al., 1955b) are present in our basal ration. The four-member factorial study reported in Table 5 indicates that the fish solubles factor is contained in liver residue. This agrees with the finding of similar factors in a liver fraction and condensed fish solubles by Combs et al. (1954) and in whole liver powder and fish meal by Tamimie (1955). The presence of lesser amounts of alfalfa-whey factor in liver residue and distillers solubles, and a relatively small amount of either distillers solubles factor or liver factor in alfalfa-whey can account for the lower growth response secured from fish solubles in relation to the other three individual supplements (Table 5). Results with two-member additions indicate that the alfalfa-whey supplement contained some liver residue factor but no appreciable amount of the distillers solubles factor. This is indicated by the somewhat lower maximum response from the alfalfa-whey plus fish solubles. The three-member supplements (Table 5) ( D D S + F S + L R a n d D D S + A W + L R ) produced definitely increased growth over the two-member additions whereas DDS + A W + F S and A W + F S + L R and the four-member supplements failed to produce expected gains. Lower caloric content in the multiple-supplemented rations was indicated and the addition of 10% fat to the four-member supplement supported this conclusion. When productive energy was not adjusted (Table 6), energy again seemed to limit growth with the two-member and three-member supplements. Better gains with equicaloric rations over those not adjusted for productive energy is most likely a further expression of unidentified factors. Increase in feed efficiency appears
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and from other trials in which half-maximum response levels of alfalfa and whey gave growth equivalent to the maximum from whey. Failure to secure an alfalfa meal response equal to whey may be due to the alfalfa inhibitor (Lepkovsky et al., 1950). The finding of a single factor in alfalfa and whey does not agree with the observations of Fisher et al. (1954) or Combs et al. (1954) and may be due to differences in the basal rations. Combs et al. (1954) indicate the presence of more than one factor in alfalfa meal. The blood meal and other natural materials used in our basal diet may contain such a second factor. Our results, however, demonstrate the presence of "whey factor" in alfalfa meal.
51
52
R. A. RASMUSSEN, P. W. LUTHY, J,. M. VAN LANEN AND C. S. BORUFF
The relationship of the factors herein reported to the grass juice factor (Norris, 1954) can only be speculative; however, as fish solubles and distillers solubles are common to both studies it is possible that the grass juice factor is similar to this study's alfalfa-whey factor. The relationship of the brewers yeast and molasses distillers solubles factors of Combs et al. (1954) to the present factors await further study. With our basal ration, a chick growth response is not obtained with molasses distillers solubles. This and the other factors mentioned
which give no response may be supplied by the basal diet or carried over from the maternal diet. With gelatin and blood meal in the basal diet it is unlikely that a response to the gelatin factor (Benton et al., 1955) would be obtained. The three unidentified factors reported here have also been demonstrated by Fisher et al. (1954) and Scott (1955). SUMMARY
An inexpensive basal diet considered to be adequate in known nutritional essentials has been devised and used in experimental chick studies of unidentified factors required by the non-depleted chick. It produces good growth and permits the separation of unidentified factors as indicated by increased growth responses with various combinations of unidentified factor carriers fed at maximum response levels. Evidence which suggests that three different unidentified factors are essential for rapid growth of chicks has been found, viz.: 1) A factor present in alfalfa meal and dried whey. 2) A factor present in Wilson's liver residue and fish solubles. 3) A factor present in distillers dried solubles. With the exception of the fish solubles each of these unidentified factor carriers appears to contain lesser amounts of the other factors. Fish solubles appears to contain only the liver residue factor. When the productive energy was maintained at the level of the basal ration (1,077 Calories per pound) the unidentified factors in the various supplements were more fully expressed. ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Virgil L. Boone and William H. McDaniels in carrying out
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to be the major effect of additional calories if a ration contains 1,000 Calories or more of P.E. per pound and more than adequate amount of other required nutrients. This increased feed efficiency may be accompanied by a small increase in weight gain. This is true in the case of liver residue (Table 6) and is indicated in the studies of Combs and Romoser (1955), wherein differences of gains of 1.6 percent are reported between 1,087 and 1,240 Calories P.E. per pound of ration while feed efficiencies were improved from 1.72 to 1.60 pounds of feed per pound gain. The energy value of a ration is a primary factor in regulating consumption of feed (Hill and Dansky, 1954; and Mellen et al., 1954). Thus decreased growth was encountered when the rations (Table 6) were equalized in their ratio of productive energy to protein, because the high calorie content of the rations decreased intake of some nutrient(s) below that required for maximum growth. For three-member supplements, over 29% of fat was required to maintain the productive energy per pound at 41 Calories per percent protein. On these diets most deaths occurred during the first week. Whether fat per se, a dietary imbalance or some other factor caused the deaths could not be ascertained.
UNIDENTIFIED GROWTH FACTORS
this study. They are indebted also to Mr. H. C. Schaefer of the Ralston-Purina Co. for the fish solubles used. The liver residue was obtained from Wilson and Company, Chicago, Illinois, and is the residue of minced whole liver extracted with acidulated water and combined with the heat-coagulated residue of the extract. REFERENCES
Further studies on the alfalfa factor and its relation to the liver and whey factors. / . Nutrition, 52:13-24. Hill, F. W., and L. M. Dansky, 1954. Studies of the energy requirements of chickens. 1. The effect of dietary energy level on growth and feed consumption. Poultry Sci. 33: 112-119. Kodras, R., W. I. Cooney and J. S. Butts, 1951a. Chick growth depressing factor in sun-cured and dehydrated alfalfa meals. Poultry Sci. 30: 280-291. Kodras, R., W. I. Cooney and J. S. Butts, 1951b. Effect of alfalfa meal, alfalfa leaves, alfalfa stems and fresh alfalfa on chick growth. Poultry Sci. 30: 768-787. Kohler, G. O., and W. R. Graham, Jr., 1951. A chick growth factor found in leafy green vegetation. Poultry Sci. 30:484-491. Lepkovsky, S., W. Shaeleff, D. Peterson and R. Perry, 1950. Alfalfa inhibitor in chick rations. Poultry Sci. 29:208-213. March, B., J. Biely and S. P. Touchburn, 1955. Studies on an unidentified chick growth factor in dehydrated green feed. Poultry Sci. 34: 968-972. Mellen, W. J., F. W. Hill and H. H. Dukes, 1954. Studies of the energy requirements of chickens. 2. Effect of dietary energy level on the basal metabolism of growing chickens. Poultry Sci. 33: 791-798. Menge, H., G. F. Combs, Peng-Tung Hsu and M. S. Shorb, 1952. Unidentified growth factors required by chicks and poults. 1. Studies with chicks using purified diets. Poultry Sci. 31: 237-247. Norris, L. C , 1954. Unidentified chick growth factors in distillers dried solubles. Proc. 8th Distillers Feed Research Conference, pp 20-28. Norris, L. C , 1955. Recent progress on unidentified growth factors in distillers dried solubles and other feed supplements. Proc. 10th Distillers Feed Research Conference, pp 39-42. Petersen, C. F., A. C. Wiese and A. R. Pappenhagen, 1955. Unidentified chick growth factors. 1. Purified assay diet and crude supplement response. Poultry Sci. 34:673-678. Rasmussen, R. A., P. W. Luthy, J. M. Van Lanen and C. S. Boruff, 1954. The dual nature of the unidentified chick growth promoting activity of distillers dried solubles. Proc. 9th Distillers Feed Research Conference, pp 29-37. Reid, B. L., R. L. Svacha, A. A. Kurnick, F. M. Salama and J. R. Couch, 1956. Unidentified growth factors and thioctic acid in chick and poult nutrition. Distillers Feed Conference 11: 68-79.
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2. THE IMMUNE RESPONSE OF CHICKENS VACCINATED WITH Bl NEWCASTLE DISEASE VIRUS ADMINISTERED THROUGH THE DRINKING WATER* R. W. WINTERFIELD AND E. H. SEADALE Department of Veterinary Science, University of Massachusetts, Amherst, Mass. (Received for publication July 9, 1956)
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HIS investigation was undertaken to further study and to determine the efficacy of mass vaccination of chickens for Newcastle disease (ND) via the drinking water (Luginbuhl et al., 1954; Winterfield and Seadale, 1955; Luginbuhl et al., 1955). Factors having a possible influence on immunity induced from this route of administration were studied, and the results are presented. MATERIALS AND METHODS
Bl strain Newcastle disease virus (NDV) (Hitchner and Johnson, 1948) vaccines, designated A, B, and C, used in all trials, were obtained from three sources. Vaccine A was a lyophilized product of commercial origin. It was supplied in 500dose vials accompanied by 15 ml. diluent per vial in separate, plastic containers. It was suggested that chicks 3 to 21 days of age be vaccinated after being deprived of water for 4 hours. The dried virus was reconstituted with the diluent and im* Contribution No. 1042 of the Massachusetts Agricultural Experiment Station.
mediately added to the drinking water in the ratio of 1:315.3. Vaccine B, also of commercial origin, was a glycerinated virus preparation supplied in 60-ml., 1,000dose quantities per bottle with an accompanying package of dried, nutrient stabilizer. One ml. of the vaccine was added to 315.3 ml. of water after a premixture had been made with 30 ml. of water and .41 gram of stabilizing material. Vaccine C represented Bl—NDV lyophilized in the authors' laboratory in 3-ml. quantities per vial from a 1:1 mixture of 10 percent skim milk and infected amnioallantoic fluid. It was reconstituted and diluted in the drinking water in the manner described with vaccine A. A stabilizer composed of two-thirds gelatin and one-third dried skim milk was added in the ratio of 1.3 grams to 315.3 ml. of drinking water. Previous studies (Winterfield and Seadale, 1956) demonstrated the ability of a stabilizer to maintain higher vaccine titers in water under simulated conditions of field administration. Single distilled water was used in all trials. Titrations of vaccines and vaccine-
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Newcastle Disease Immunization Studies