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The Effects of Arsanilic Acid on Laying Hens at Three Dietary Protein Levels1
RETICULOCYTE COUNTS The response of grain-fed pigeons to substances effective in pernicious anaemia. Brit. J. Exp. Path. 1 1 : 456-468.
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Wills, L., 1932. Spontaneous fluctuations in the reticulocyte counts in pigeon's blood. Brit. J. Exp. Path. 13 : 172-175.
The Effects of Arsanilic Acid on Laying Hens At Three Dietary Protein Levels1 PART 2. FERTILITY, HATCHABILITY, CHICK GROWTH AND BLOOD SPOT INCIDENCE D. K. ANDREWS,2 H. R. BIRD AND M. L. SUNDE Department of Poultry Science, University of Wisconsin, Madison, Wisconsin (Received for publication April 4, 1966)
E
ARLIER investigation by Libby et al. (1953) determined the presence of residual arsenic in the egg to be of little consequence and well below the limits allowed by the Food and Drug Administration. Neither they nor Wharton and Fritz (1953) found any significant effects of the arsonic acids on hatchability or early progeny growth. Lillie et al. (1957) concluded after 3 years of experiments with dual purpose breeds that arsanilic acid alone or in combination with fat and antibiotics in 15% protein diets did not influence fertility or hatchability but some positive maternal diet influence was noted in the chicks. Carlson (1957) adding 90 gms. of arsanilic acid per ton to 12, 15 and 16% protein rations reported improved production and hatachability but found no consistent effects on progeny growth. Later, Carlson (1959) continued the life cycle experiments with small numbers of chicks with beneficial but still inconclusive results. Kurnick et al. (1960) feeding 17% protein diets and arsanilic acid recorded
improved growth at 48 days of age in progeny from dams fed arsanilic acid although fertility or hatchability were unaffected.
1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Present address: Extension Poultry Specialist, Western Washington Experiment Station, Puyallup, Washington. This work was supported, in part, by a grant from Abbott Laboratories, North Chicago, Illinois.
RESULTS AND DISCUSSION
EXPERIMENTAL PROCEDURE
General management and procedure were similar to those discussed by Andrews et al. (1966). Fertility experiments were primarily conducted with the cage birds using weekly inseminations of .05 cc. of pooled semen from caged New Hampshire males. As a further check, 2 experiments were conducted with artificial insemination of the floor hens by the same males. Fertility was measured by breaking out all undeveloped eggs at 7 days and examining the germinal disc to see if life had started. Additional breakouts were made at the 22nd day of incubation. Two groups of chick experiments were conducted. Hatch 1 and hatch 2 were from the first experiment while hatches 3, 4, and 5 were from the second one. All chicks were pedigree hatched, grouped in lots and reared in electric chick batteries.
Fertility. Eggs from the cage treatments were set during the winters of both experiments. Floor pen eggs were set from February to April of the second experiment. Arsanilic acid treated hens, at all dietary
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE TABLE 1.—Fertility and hatchability of groups fed arsanilic acid, and control diets at three dietary protein Percent hatch of total eggs
Percent protein diet2
Percent fertile eggs
Cage
Cage
Floor
Exp. 1
Exp. 2
Exp. 2
12% 12%AA 12%AAAB
84.0 84.8 85.0
73.6 74.1 78.5
14% 14%AA 14%AB 14%AAAB
81.8 80.8 82.7 78.5
16% 16%AA
84.5 87.6
Cage
Floor
Exp. 1
Exp. 2
Exp. 2
57.6 86.7***
91.5 97.2* 97.3
88.2 92.7 97.8
74.6 95.6***
57.8 72.1*** 65.4 65.8
58.9 66.1
96.0 97.9 96.3 93.9
92.0 95.6 92.4 90.8
72.9 90.1***
67.6 75 4***
63.7 70.2
92.2 99 5***
88.7 93.8*
75.0 86.6**
* Statistically significant at .05 level. ** Statistically significant at .01 level. *** Statistically significant at .005 level. 1 Exp. 1 cage hens—3 hatches—2,054 eggs—1693 chicks—82.4%. Exp. 2 cage hens—4 hatches—3,067 eggs—2146 chicks—70.0%. Exp. 2 floor hens—2 hatches—1,450 eggs—974 chicks—67.2%. 2 Abbreviations used: AA = arsanilic acid, AB = antibiotic, AAAB = both AA and AB.
protein levels, had rather consistently better fertility than their controls (Table 1). This observation is in agreement with Carlson (1957) and Barnett, Richey and Morgan (1959) but opposed to Libby et al. (1953), Lillie et al. (1957) and Thornton and Moreng (1958) who noted no fertility differences in their experiments. The protein level of the control diets had no effect upon fertility or hatchability. Among the separate hatches arsanilic acid improved fertility 18 times, about equal 6 times and 2 times was lower than the control group. Probability was determined for the data, pooled by experiments and environment, for each of the 3 protein levels, by using adjusted y.2 I 0 r inter-action and normal approximation (Steel and Torrie, 1960). Arsanilic acid statistically improved fertility (P <.05) 3 times out of 6 among the cage birds and every time in the floor pens ( P < . 0 0 5 twice and P <.01). Percent hatch. Summarization and y? treatment of the hatchability data results, while not as consistant as those shown by the
fertility data, demonstrate that arsanilic acid increased percent hatch 17 times, had no effect once and had negative effects 8 times out of the 26 treatments. Percent hatch of the total eggs set was significantly improved (P <.005) by arsanilic acid in the second experiment at the 14 and 16% protein levels of the cage birds and (P <.05) at the 12% protein level of the floor birds. During the first experiment hatchability remained in the 80% range and little difference was noted (Table 1). During the second experiment a generally poorer hatch was experienced. This may indicate the value of arsanilic acid in flocks where fertility and percent hatch is a problem. Since percent hatch also includes fertility, it was obvious that the greatest effect of arsanilic acid was on the fertility. When the averages of all the percent hatchability figures (percent hatch of fertile eggs) were determined the ones without arsanilic acid showed a hatchability figure of 81.6% and the ones with arsanilic acid a figure of 82.1%. This difference does not approach significance.
ARSANILIC ACID IN LAYING RATIONS
Chick growth. Frost (1953) reported that eggs from hens fed for 5 months on good laying diets containing 90 to 270 gms./ton of arsanilic acid were found to contain an arsenic equivalent of about 0.5 p.p.m. As203 as against 0.03 p.p.m. for the control. No difference was noted between the 2 widely different feeding levels as to the arsenic retention in the eggs. Chicks from the first experiment were each divided in 2 groups. One half received an 18% and the other a 23% protein chick starter (Table 2). Chicks from hatch 2 were sexed and weighed at hatching while those from hatch 1 were not. Very slight weight increases were recorded at the 1, 2, and 4-week weights in favor of the chicks from arsanilic acid fed dams. A comparison of the average weekly weights of the 1215 chicks was as follows: 1 week, 14 lots increased in weight, 4 decreased, 2 were the same; 2 week, 16 lots increased in weight, 3 decreased, 1 was the same; 4 week, 12 lots increased in weight, 7 decreased, 1 was the same. Chicks fed the 23% protein diet made TABLE 2.—Percent composition of all
mash chick starter diets Ingredients Ground yellow corn Corn gluten meal Wheat middlings Soybean meal (44% protein) Distillers corn solubles Alfalfa meal, dehydrated Fish meal Dried whey Steamed bone meal Limestone Grease (choice-white) A and D oil (300 D 800 A/gm.) Methionine hydroxy analog Manganese sulfate Salt Vitamins (added as gms./45 kg.) Riboflavin Niacin Ca. pantothenate Choline (70%) Bi! (9 mg./454 gms.) Determined analysis % protein Calories (Productive energy/454 gms.)
Percent Protein of Diets 18%
23%
65.0 3.5 4.2 13.0 2.0 4.0 2.5 2.0 1.7 0.8 0.4 0.2 0.1 .012 0.4
45.0 5.0 5.0 25.0 2.5 5.0 4.0 2.5 2.0 1.0 2.0 0.2 0.1 .012 0.5
0.1 0.5 0.3 80.0 25.2 18.4 945
0.1 0.5 0.3 80.0 25.2 23.8 871
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progressively more rapid weight gains each week than those fed the 18% protein diet. Hatches 3 and 4 from the second experiment were sexed, weighed and arranged in chick batteries in groups by sex and dams' diet for the purpose of obtaining feed conversion as well as growth data. To increase the number of chicks available, hatch 4 contained chicks from the floor hens as well as the cages. Five hundred eighty (580) chicks from hatch 3 and 800 chicks from hatch 4 were divided into 10-chick lots. All chicks received the 23% protein chick diet. Small but consistent weight gains were again demonstrated by the chicks as given in the following comparisons. 1 week, 13 lots increased in weight, 7 decreased; 2 week, 16 lots increased in weight, 3 decreased and 1 was the same; 4 week, 14 lots increased in weight, 5 decreased and 1 was the same. Feed conversions of hatches 3 and 4 indicate slight improvement in feed utilization by 19 of the 24 comparisons of both male and female progeny of the dams fed 12% protein diets supplemented with arsanilic acid. Neither the 14 nor 16% protein diets experienced any difference. Hatch 5 was utilized to determine possible additive effects that might be obtained by feeding a .02% arsanilic acid supplemented diet to progeny of dams also fed .02% arsanilic acid. Results shown in Table 3 demonstrate that the effects of adding .02% arsanilic acid supplement to the dams' diet was again manifested in increased early chick growth and, furthermore, this maternal effect was present over and above the growth improvement noted when arsanilic acid was added to the chicks' diet. Chicks from dams fed arsanilic acid again outweighed their respective control groups (23 increased in weight, 8 de-
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
TABLE 3.—Effects
ofmaternal diet on chick weight of progeny1 fed diets with and without .02% arsanilic acid Group average chick weights in grams
Maternal diet % protein2
Chick control diet Both sexes
Male
1 wk.
2 wks.
12% 12%AA 12%AAAB
72.4 78.2 74.3
142 142 139
363 355 358
14% 14%AA
71.9 75.3
139 141
16% 16%AA
70.9 74.1
Control AA&AAAB
71.7 75.5
Chick diet+arsanilic acid Female
4 wks.
Both sexes
Male
Female
1 wk.
2 wks.
4 wks
342 326 331
72.8 76.8 75.6
142 146 150
360 376 382
320 329 328
340 366
327 334
75.6 77.2
146 148
391 380
340 344
136 142
358 370
328 333
71.8 80.0
141 148
377 372
338 336
139 141
354 362
332 331
73.4 77.4
143 148
376 378
333 334
Summary: Additive effects of arsanilic acid in dam and progeny diets Hen and chick diets AA C AA AA
hen hen hen hen
.
& C chix against C hen & C Chix & AA chix against C hen & C chix & AA chix against C hen & AA chix & AA chix against C hen & C chix
1 wk.
2 wks.
4 wk. M
+3.8 + 1.7 +4.0 +5.7
+2 +4 +5 +9
+8 +22 +2 +24
4 wk. F. -1
+1 +1 +2
1
449 chicks. Abbreviations used: AA=arsanilic acid, AB = antibiotic, AAAB = AA+AB, C = control diets, Chix = chickens, M = male, F = female. 2
creased and 1 weighed the same). When the progeny of dams fed arsanilic acid supplement were themselves fed .02 % arsanilic acid they increased body weight 11 out of 12 times over chicks fed the same diet but from dams without arsanilic acid treatment. Vitamin K deficiency symptoms. Observations in late September and early October (Experiment 2) revealed that chicks from the cage layers now entering their second year of lay were bleeding after wing banding. For the next 5 months all chicks from the cage groups were pedigree hatched and wing banded. Hemorrhages were scored the following day. Diets were calculated and found to contain the required 0.5 mg./kg. of menadione. Review of mixing procedure eliminated any reasonable chance that vita-
min K had been omitted from the premix. The vitamin premix in question was then approximately 4 months old but had been stored under refrigeration. As an initial check that a vitamin K deficiency existed, 12 hens with the highest number of afflicted chicks were given 2 intraperitoneal injections of 5 mgs. each of menadione sodium bisulfite in \ cc. of water. Injections were made on October 30 and November 9. Hemorrhages ceased immediately after these injections, while the controls continued with the same high incidence. It should be noted that at no time did the hens exhibit any evidence of improper diets and this vitamin deficiency would not have been noticed if the chicks that hatched had not been wing banded. This effect had been noted by Cravens et at. (1941).
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ARSANILIC ACID IN LAYING RATIONS 50 NEW PREMIX ADDED
. tt
MENADIONE SODIUM B I S U L F I T E
ADDED
^o •
/
\
S\.
30
20
9
10
*
V 1
10/11
10/25
1
-1
11/8
11/22
^ > 1
12/6
1
12/20
1
1/3
1/17
,. 1
1
\
V^N
1 —
*_l
1/31
2/14
2/28
3/7
WEEKLY HATCH DATES
FIG. 1. Incidence of wing hemorrhages induced by banding chicks hatched from vitamin K deficient dams.
Chick wing hemorrhages were diagnosed depending upon the degree of bleeding into 4 classifications: death (D), severe (S), intermediate (I), and small (s). During the early observations, an estimated onehalf of the hemorrhages were (S) or (I). This decreased to a small amount during the last hatches. A new vitamin premix (same formula, same menadione) was utilized during the last of October. Results of the new vitamin premix (Fig. 1) indicate an immediate reduction from 30% wing hemorrhages to as low as 10% wing hemorrhages among the chicks during the next 3 weeks. The incidence rose again to 25% bleeders and then slowly decreased to 9%. Menadione sodium bisulfite supplement (1.0 mg./kg., atomic weight 330) was substituted for menadione (atomic weight 172) and chicks from this combination were hatched in early February. By March 8, all chicks were normal. The 12% protein diets were not as severely affected as the 14 and 16% protein diets during the first 10-week period of observation (Table 4). During the last 8 weeks there was no difference in bleeding.
The arsanilic acid supplemental groups were detrimentally affected. At the end of the first 10-week summary the control groups were experiencing 12, 28, and 26% incidence of bleeding while the comparable arsanilic acid groups had 21, SO and 35% wing hemorrhage. The last 8-week summary (January 3 to March 7) shows the control group averages to be 2, 3 and 7% bleeders while the arsanilic acid groups were 12, 14, and 4% bleeders respectively. By March 7 all groups were fully recovered. Although these findings were obtained
TABLE 4.—Percentage of vitamin K deficient cage
hens' of spring suffering wing band bleeding Diet percent1 protein
Chick hatching dates Oct. 18, March 8, (experiment 2) Summary 10 hatches to 12/20
Summary 8 hatches to 3/7
12% 12%AA 12%AAAB
12 21 22
2 12 8
1,213/181 (14.9%)
14% 14%AA 14%AB 14%AAAB
28 50 31 32
3 14 8 12
1.406/362 (25.7%)
16% 16%AA
26 35
7 4
726/148 (20.4%)
1
No. eggs/bleeders
Abbreviations: AA=arsanilic acid 0.02%, AB =aureomycin 20 gms./ton, AAAB =both AA and AB.
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
with the breeding hen fed a diet supplemented with .02 % arsanilic acid, they substantiate the reports of Griminger et al. (1953) who noted a significant increase (P < .05) in blood clotting time in 4 week old chicks fed .01% arsanilic acid. Menadione (5 mgs./kg.) significantly reduced blood clotting time in the controls and among the arsanilic acid fed chicks. Griminger (1962) repeating the experiment again concluded that arsanilic acid modified the intestinal synthesis of vitamin K, but was not antagonistictoit. Chicks from antibiotic fed dams or antibiotic and arsanilic acid supplemented dams were intermediate in response between the arsanilic acid treatments and the controls. This was in agreement with Nelson and Norris (1959). Review of the individual hens with large
numbers of chicks having wing hemorrhages as well as hens having chicks with no wing hemorrhages reveals no relationship with the incidence of blood spots in eggs. While vitamin K was involved with the wing hemorrhages in the banded baby chicks it apparently was not related to the blood spot incidence in the eggs as the blood spot frequency (Table 5) was still increasing in January when the chick hemorrhages were greatly decreased. This observation would be in agreement with Perdue et al. (1961) who noted no relationship between vitamin K and blood spot incidence. Blood spot incidence. Nine observations were made of the cage birds' eggs starting with the initial pre-experimental period. Later, examination was made of samples of
TABLE 5.—Percent blood spot occurrence in cage layers fed arsanilic acid and antibiotic at three dietary protein levels Experimental periods (experiment 2)
Pre exp. Oct.
Dec.
Jan.
May
Oct.
Nov.
Dec.
Jan.
12% 12%AA 12%AAAB Average
0 2.4 0 0.8
0 0 0 0
0 3.3 0 1.0
0 0 0 0
4.2 4.8 0 3.0
8.0 0 3.8 4.0
5.0 7.3 5.0 5.8
3.7 3.6 0 2.7
14% 14%AA 14%AB 14%AAAB Average
0 4.6 2.5 0 1.8
10.0 10.5 0 0 4.9
0 12.8 2.6 2.7 4.6
0 14.3 0 0 3.9
0 0 0 0 0
0 3.6 7.7 0 2.7
6.3 15.6 3.3 4.8 7.5
2.5 23.8 0 0 4.2
16% 16%AA Average
5.1 4.4 4.8
0 5 2.4
0 13.9 6.8
5 8.3 6.6
0 16.7 7.7
4.0 33.3 16.3
8.5 27.0 15.9
19.2 31.6 24.4
Grand Ave.
2.1
3.0
4.0
3.4
2.6
5.7
8.7
7.6
ooo
Diet percent protein 1
0 0 4.0
6.8 12.6 3.4
4.0 11.8 9.1
Adjusted data 3
12%AA 14%AA4 16%6 1
0 0 2.8
0 6.7 0
3.7 12.9 0
0 11.4 2.7
2
Abbreviations used: AA=arsanilic acid, AB = antibiotic, AAAB = both AA and AB. Adjusted data corrected by removal of hens whose eggs contained blood spots during control period and more than 1 occurrence later. 3 Removed hen B 26 (29 eggs, 3 blood spots). 4 Removed hen F 15 and F 5 (47 eggs, 13 blood spots). 6 Removed hen H 102 (22 eggs, 10 blood spots). 2
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ARSANILIC ACID IN LAYING RATIONS TABLE 6.—Blood spot incidence in cage layers fed arsanilic acid and antibiotic at three protein levels {Adjusted data)1 Experiment 2 Percent protein diet2
Pre exp. diet Hens affected
Hens affected3
HensX breakout periods4
12% Control 12%AA 12%AAAB Total
-0-0-0-0-
3/12 3/12 4/12 10/36
6/96 5/96 5/96 16/288
14% Control 14%AA 14%AB 14%AAAB Total
-0-01/12 -01/48
5/12 7/12 3/12 5/12 20/48
9/96 14/96 N S 6/96 5/96 34/384
16% Control 16%AA Total
1/12 2/12 3/24
5/12 7/12 12/24
7/96 22/96*** 29/192
Eggs/Blood spot No. 223/8 214/7 232/5 669/20 281/9 223/21 275/6 246/5 1,025/41 245/7 210/41 455/48
% 3.59 3.27 2.15 2.98 .20 41* .18 ,03 .00 2.86 21.90*** 10.55
** Significant at .01 level.6 *** Significant at .005 level. Adjusted data corrected by removal of 4 birds whose eggs contained blood spots during pre-experiment period and more than 1 occurrence during experiment. 2 Abbreviations: AA=arsanilic acid, AB = antibiotic, AAAB = AA and AB. 3 Additional hens laying blood spots. Does not include blood spot layers during pre-experiment period. 4 Number of hens in the group laying one or more bloodspot eggs during each breakout period. 6 Determined by x 2 f ° r interaction and normal approximation (Steel and Torrie, 1960). 1
eggs laid by the sibs of these birds maintained in floor pens. Breakout periods were extended to as long as 20 days to reduce variation which occurred during the earlier 3-day breakout periods. All blood spots were recorded. These varied from pin point size to large masses although the latter were few. Standard breakout equipment was used and all eggs were at least 24 hours old. Summarization of the pre-experimental data (taken when the birds first started laying) by percent based upon the individual hens (Table 5) indicated that 8 of the 108 birds laid an egg containing 1 or more blood spots during the pre-test period of 5 days. Two of these hens laid no further blood spots, 2 laid 1 each. The remaining 4 hens were removed from the analysis. Table 6 excludes the 4 bloodspot prone birds. The column headed Hens Times Breakout Periods considers the persistency with which a hen laid blood spot eggs dur-
ing the 8 separate breakout periods. Observations based upon the control groups (Table 6) indicate that the persistency (6/96, 9/96, 7/96) and intensity (3.6%, 3.2%, and 2.9%) with which the hens continued to lay blood spots remained approximately the same at all protein levels. These data were found to be in disagreement with Bearse et al. (1962) who reported a positive association between blood spot incidence and increased dietary protein levels of 14, 16, and 18%. Upon the inclusion of .02% arsanilic acid the 14 and 16% protein diets appeared to increase the probability of a hen to lay one or more blood spots during her lifetime (12% diet 5/96, 14% diet 14/96, and 16% diet 22/96). The 16% protein diet supplemented with arsanilic acid was significantly greater (P < .005) than the control group in the numbers of hens and the persistence with which they continued to lay blood spot eggs. Among these cage layers, arsanilic acid,
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
while having no effect at the 12% protein level, produced significantly higher numbers of blood spots (P <.005) at the 14 and 16% protein levels as determined by adjusted x2. Omitting the 4 questionable hens changed the "/2 values only slightly, causing the 14% adjusted group comparison to drop to P <.01. Treatments containing 20 grams per ton of antibiotic alone and with arsanilic acid had the lowest blood spot incidence (2% and 2.2%) in the 12 and 14% protein groups. This beneficial response to chlortetracycline is contrary to the reports of other research workers. Chin and Brant (1953), Bearse and Berg (1955), and White-Stevens (1963) report no difference. A comparison of the U.S.D.A. Egg Production Tests summary data (1961, 1962) substantiates the results found at this station. Six of the Random Sample Test rations had an antibiotic supplement and 6 had no antibiotic. Blood spot incidence among the 6 without antibiotic supplement was. 4.9% the 1st year and 5.3% the 2nd year. Those with antibiotic supplement had 3.3 and 3.1% incidence of blood spots. Correspondence confirmed that no changes had occurred in the diets. Inspection of the 1963 and 1964 reports reveals that the same relationship continued. Daily blood spot incidence and frequency varied greatly, fluctuating from 0 to 10% and 0 to 33%, by periods (Table 5). Seasonal differences were noticeable and appear to have increased during the 2nd year of lay. A comparison of cage layers to their floor pen sisters on identical diets indicates substantially the same information as that found among the cage birds. Protein level was not associated with blood spot incidence. The 16% protein diets with arsanilic acid appeared to cause a significantly (P >.05) higher blood spot occurrence than was found in the control diet. Floor
pen control and adjusted battery controls had approximately the same percentages (12% diet 3.2-3.6; 14% diet 4.2-3.2; and 16% diet 2.2-2.9). Hens in floor pens fed arsanilic acid appeared to lay about the same number of blood spot eggs at all protein levels (12% diet 6.5%; 14% diet 4.8%; and 16% diet 5.8%). SUMMARY
Two years' observations of 12, 14, and 16% protein laying diets with and without .02% arsanilic acid in both cages and floor treatment show that: 1. Fertility and total number of chicks hatched were statistically improved by the addition of arsanilic acid. 2. The progeny from hens fed arsanilic acid exhibited slight growth increases over chicks from dams on corresponding control diets. This maternal effect was present over and above that found by feeding the chicks a .02% arsanilic acid supplemented diet. 3. Cage birds receiving 14 and 16% protein diets with .02% arsanilic acid increased significantly in blood spot incidence. To a lesser degree this was also true in the floor pens at all protein levels. 4. Hens fed .02% arsanilic acid diets had a higher minimal vitamin K. requirement than sibs on corresponding control diets. REFERENCES Andrews, D. K., H. R. Bird and M. L. Sunde, 1966. The effects of arsanilic acid on laying hens at three dietary protein levels. Part I. Egg production. Poultry Sci. 45: 838-847. Barnett, B. D., D. J. Richey and C. L. Morgan, 1959. The effect of feeding terramycin, furazolidone, and arsenicals to breeder hens. Assoc, of Southern Agr. Workers, Memphis, Tenn. p. 237. Bearse, G. E., and L. R. Berg, 1955. The effect of varying levels of aureomycin on the performance of young laying chickens. Poultry Sci. 34 : 1180. Bearse, G. E., L. R. Berg and A. H. Massey, 1962.
ARSANILIC ACID IN LAYING RATIONS Protein level of the diet and incidence of blood spots in chicken eggs. Poultry Sci. 4 1 : 1625. Carlson, C. W., 1957. Some effects of arsanilic acid and/or penicillin upon egg production. Poultry Sci. 36: 1070-1075. Carlson, C. W., 1959. Effects of antibacterial and antifungal agents in diets for laying and breeding hens. South Dakota Agr. Sta. Tech. Bui. No. 22: 1-23. Chin, G., and A. W. Brant, 1953. Egg quality and aureomycin. Poultry Sci. 32: 875-876. Cravens, W. W., S. B. Randle, C. A. Elvehjem and J. G. Halpin, 1941. Vitamin K studies 1. Effect of the vitamin K content of the hen's ration on the clotting ability of chick blood. Poultry Sci. 20: 313-316. Frost, D. V., 1953. Considerations on the safety of arsanilic acid for use in poultry feeds. Poultry Sci. 32: 217-225. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953. Factors influencing blood clotting time in the chick. Science, 118: 379-380. Griminger, P., 1962. Arsanilic acid and blood coagulation. Poultry Sci. 4 1 : 982-985. Kurnick, A. A., R. J. Pforsich, M. W. Pasvogel, H. B. Hinds and L. W. Dewhurst, 1960. Effects of furazolidone and arsanilic acid on the reproductive performance of hens under conditions of varying temperatures. Proceedings Third National Symposium on Nitrofurans. p. 136-150. Libby, D. A., A. C. Groschke, R. J. Evans and
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S. L. Bandemer, 1953. Feeding of arsanilic acid to pullets. Michigan Agr. Exp. Sta. Quart. Bui. 35: 419-427. Lillie, R. J., J. R. Sizemore and C. A. Denton, 1957. Effect of an arsenical, fat, and antibiotic upon the reproductive performance of chickens. Poultry Sci. 36: 755-759. Nelson, T. S., and L. C. Norris, 1959. Factors affecting the vitamin K requirement of the chick. Poultry Sci. 38: 1094-1102. Perdue, H. S., J. A. Kolar and D. V. Frost, 1961. Non-correlation of vitamin K status and production of eggs with blood spots. Poultry Sci. 40:1443. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, N.Y. Thornton, P. A., and R. E. Moreng, 1958. Egg production and reproduction as affected by arsanilic acid and penicillin. Poultry Sci. 3 7 : 554557. United States Department of Agriculture, 1961. Report of egg production tests for the period 1960-1961. ARS 44-79-2. United States Department of Agriculture, 1962. Report of egg production tests for the period 1961-1962. ARS 44-79-3. Wharton, F. D., Jr., and J. C. Fritz, 1953. Studies on the effect of an arsenical in the diet of the immature and mature chicken. Poultry Sci. 32 : 1014-1020. White-Stevens, R. H., 1963. Personal Communication. American Cyanamid Co., Princeton, N.J.
Effect of Increasing Dietary Levels of Acetyl-Salicyhc Acid on Performance and Cecal Microbial Counts of White Leghorn Pullets1 J. M. THOMAS,2 H. S. NAKAUE AND B. L. REID Poultry Science Departent, University of Arizona, Tucson, Arizona (Received for publication April 7, 1966) INTRODUCTION
AN earlier report from this station, Reid A et al. (1964), showed that when 0.05% acetyl-salicylic acid (ASA) was fed 1
Arizona Agricutural Experiment Station Technical Paper No. 1111. 2 Present address: Batelle 'Memorial Institute, Richland, Washington.
White Leghorn pullets either alone or in combination with 27.5 mg. oxytetracycline per kg., a significant increase in egg production resulted. The improvement was independent of periods of high ambient temperature and thus the action of the drug was assumed to be exclusive of its antipyretic properties.
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Wills, L., 1932. Spontaneous fluctuations in the reticulocyte counts in pigeon's blood. Brit. J. Exp. Path. 13 : 172-175.
The Effects of Arsanilic Acid on Laying Hens At Three Dietary Protein Levels1 PART 2. FERTILITY, HATCHABILITY, CHICK GROWTH AND BLOOD SPOT INCIDENCE D. K. ANDREWS,2 H. R. BIRD AND M. L. SUNDE Department of Poultry Science, University of Wisconsin, Madison, Wisconsin (Received for publication April 4, 1966)
E
ARLIER investigation by Libby et al. (1953) determined the presence of residual arsenic in the egg to be of little consequence and well below the limits allowed by the Food and Drug Administration. Neither they nor Wharton and Fritz (1953) found any significant effects of the arsonic acids on hatchability or early progeny growth. Lillie et al. (1957) concluded after 3 years of experiments with dual purpose breeds that arsanilic acid alone or in combination with fat and antibiotics in 15% protein diets did not influence fertility or hatchability but some positive maternal diet influence was noted in the chicks. Carlson (1957) adding 90 gms. of arsanilic acid per ton to 12, 15 and 16% protein rations reported improved production and hatachability but found no consistent effects on progeny growth. Later, Carlson (1959) continued the life cycle experiments with small numbers of chicks with beneficial but still inconclusive results. Kurnick et al. (1960) feeding 17% protein diets and arsanilic acid recorded
improved growth at 48 days of age in progeny from dams fed arsanilic acid although fertility or hatchability were unaffected.
1 Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. 2 Present address: Extension Poultry Specialist, Western Washington Experiment Station, Puyallup, Washington. This work was supported, in part, by a grant from Abbott Laboratories, North Chicago, Illinois.
RESULTS AND DISCUSSION
EXPERIMENTAL PROCEDURE
General management and procedure were similar to those discussed by Andrews et al. (1966). Fertility experiments were primarily conducted with the cage birds using weekly inseminations of .05 cc. of pooled semen from caged New Hampshire males. As a further check, 2 experiments were conducted with artificial insemination of the floor hens by the same males. Fertility was measured by breaking out all undeveloped eggs at 7 days and examining the germinal disc to see if life had started. Additional breakouts were made at the 22nd day of incubation. Two groups of chick experiments were conducted. Hatch 1 and hatch 2 were from the first experiment while hatches 3, 4, and 5 were from the second one. All chicks were pedigree hatched, grouped in lots and reared in electric chick batteries.
Fertility. Eggs from the cage treatments were set during the winters of both experiments. Floor pen eggs were set from February to April of the second experiment. Arsanilic acid treated hens, at all dietary
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE TABLE 1.—Fertility and hatchability of groups fed arsanilic acid, and control diets at three dietary protein Percent hatch of total eggs
Percent protein diet2
Percent fertile eggs
Cage
Cage
Floor
Exp. 1
Exp. 2
Exp. 2
12% 12%AA 12%AAAB
84.0 84.8 85.0
73.6 74.1 78.5
14% 14%AA 14%AB 14%AAAB
81.8 80.8 82.7 78.5
16% 16%AA
84.5 87.6
Cage
Floor
Exp. 1
Exp. 2
Exp. 2
57.6 86.7***
91.5 97.2* 97.3
88.2 92.7 97.8
74.6 95.6***
57.8 72.1*** 65.4 65.8
58.9 66.1
96.0 97.9 96.3 93.9
92.0 95.6 92.4 90.8
72.9 90.1***
67.6 75 4***
63.7 70.2
92.2 99 5***
88.7 93.8*
75.0 86.6**
* Statistically significant at .05 level. ** Statistically significant at .01 level. *** Statistically significant at .005 level. 1 Exp. 1 cage hens—3 hatches—2,054 eggs—1693 chicks—82.4%. Exp. 2 cage hens—4 hatches—3,067 eggs—2146 chicks—70.0%. Exp. 2 floor hens—2 hatches—1,450 eggs—974 chicks—67.2%. 2 Abbreviations used: AA = arsanilic acid, AB = antibiotic, AAAB = both AA and AB.
protein levels, had rather consistently better fertility than their controls (Table 1). This observation is in agreement with Carlson (1957) and Barnett, Richey and Morgan (1959) but opposed to Libby et al. (1953), Lillie et al. (1957) and Thornton and Moreng (1958) who noted no fertility differences in their experiments. The protein level of the control diets had no effect upon fertility or hatchability. Among the separate hatches arsanilic acid improved fertility 18 times, about equal 6 times and 2 times was lower than the control group. Probability was determined for the data, pooled by experiments and environment, for each of the 3 protein levels, by using adjusted y.2 I 0 r inter-action and normal approximation (Steel and Torrie, 1960). Arsanilic acid statistically improved fertility (P <.05) 3 times out of 6 among the cage birds and every time in the floor pens ( P < . 0 0 5 twice and P <.01). Percent hatch. Summarization and y? treatment of the hatchability data results, while not as consistant as those shown by the
fertility data, demonstrate that arsanilic acid increased percent hatch 17 times, had no effect once and had negative effects 8 times out of the 26 treatments. Percent hatch of the total eggs set was significantly improved (P <.005) by arsanilic acid in the second experiment at the 14 and 16% protein levels of the cage birds and (P <.05) at the 12% protein level of the floor birds. During the first experiment hatchability remained in the 80% range and little difference was noted (Table 1). During the second experiment a generally poorer hatch was experienced. This may indicate the value of arsanilic acid in flocks where fertility and percent hatch is a problem. Since percent hatch also includes fertility, it was obvious that the greatest effect of arsanilic acid was on the fertility. When the averages of all the percent hatchability figures (percent hatch of fertile eggs) were determined the ones without arsanilic acid showed a hatchability figure of 81.6% and the ones with arsanilic acid a figure of 82.1%. This difference does not approach significance.
ARSANILIC ACID IN LAYING RATIONS
Chick growth. Frost (1953) reported that eggs from hens fed for 5 months on good laying diets containing 90 to 270 gms./ton of arsanilic acid were found to contain an arsenic equivalent of about 0.5 p.p.m. As203 as against 0.03 p.p.m. for the control. No difference was noted between the 2 widely different feeding levels as to the arsenic retention in the eggs. Chicks from the first experiment were each divided in 2 groups. One half received an 18% and the other a 23% protein chick starter (Table 2). Chicks from hatch 2 were sexed and weighed at hatching while those from hatch 1 were not. Very slight weight increases were recorded at the 1, 2, and 4-week weights in favor of the chicks from arsanilic acid fed dams. A comparison of the average weekly weights of the 1215 chicks was as follows: 1 week, 14 lots increased in weight, 4 decreased, 2 were the same; 2 week, 16 lots increased in weight, 3 decreased, 1 was the same; 4 week, 12 lots increased in weight, 7 decreased, 1 was the same. Chicks fed the 23% protein diet made TABLE 2.—Percent composition of all
mash chick starter diets Ingredients Ground yellow corn Corn gluten meal Wheat middlings Soybean meal (44% protein) Distillers corn solubles Alfalfa meal, dehydrated Fish meal Dried whey Steamed bone meal Limestone Grease (choice-white) A and D oil (300 D 800 A/gm.) Methionine hydroxy analog Manganese sulfate Salt Vitamins (added as gms./45 kg.) Riboflavin Niacin Ca. pantothenate Choline (70%) Bi! (9 mg./454 gms.) Determined analysis % protein Calories (Productive energy/454 gms.)
Percent Protein of Diets 18%
23%
65.0 3.5 4.2 13.0 2.0 4.0 2.5 2.0 1.7 0.8 0.4 0.2 0.1 .012 0.4
45.0 5.0 5.0 25.0 2.5 5.0 4.0 2.5 2.0 1.0 2.0 0.2 0.1 .012 0.5
0.1 0.5 0.3 80.0 25.2 18.4 945
0.1 0.5 0.3 80.0 25.2 23.8 871
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progressively more rapid weight gains each week than those fed the 18% protein diet. Hatches 3 and 4 from the second experiment were sexed, weighed and arranged in chick batteries in groups by sex and dams' diet for the purpose of obtaining feed conversion as well as growth data. To increase the number of chicks available, hatch 4 contained chicks from the floor hens as well as the cages. Five hundred eighty (580) chicks from hatch 3 and 800 chicks from hatch 4 were divided into 10-chick lots. All chicks received the 23% protein chick diet. Small but consistent weight gains were again demonstrated by the chicks as given in the following comparisons. 1 week, 13 lots increased in weight, 7 decreased; 2 week, 16 lots increased in weight, 3 decreased and 1 was the same; 4 week, 14 lots increased in weight, 5 decreased and 1 was the same. Feed conversions of hatches 3 and 4 indicate slight improvement in feed utilization by 19 of the 24 comparisons of both male and female progeny of the dams fed 12% protein diets supplemented with arsanilic acid. Neither the 14 nor 16% protein diets experienced any difference. Hatch 5 was utilized to determine possible additive effects that might be obtained by feeding a .02% arsanilic acid supplemented diet to progeny of dams also fed .02% arsanilic acid. Results shown in Table 3 demonstrate that the effects of adding .02% arsanilic acid supplement to the dams' diet was again manifested in increased early chick growth and, furthermore, this maternal effect was present over and above the growth improvement noted when arsanilic acid was added to the chicks' diet. Chicks from dams fed arsanilic acid again outweighed their respective control groups (23 increased in weight, 8 de-
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
TABLE 3.—Effects
ofmaternal diet on chick weight of progeny1 fed diets with and without .02% arsanilic acid Group average chick weights in grams
Maternal diet % protein2
Chick control diet Both sexes
Male
1 wk.
2 wks.
12% 12%AA 12%AAAB
72.4 78.2 74.3
142 142 139
363 355 358
14% 14%AA
71.9 75.3
139 141
16% 16%AA
70.9 74.1
Control AA&AAAB
71.7 75.5
Chick diet+arsanilic acid Female
4 wks.
Both sexes
Male
Female
1 wk.
2 wks.
4 wks
342 326 331
72.8 76.8 75.6
142 146 150
360 376 382
320 329 328
340 366
327 334
75.6 77.2
146 148
391 380
340 344
136 142
358 370
328 333
71.8 80.0
141 148
377 372
338 336
139 141
354 362
332 331
73.4 77.4
143 148
376 378
333 334
Summary: Additive effects of arsanilic acid in dam and progeny diets Hen and chick diets AA C AA AA
hen hen hen hen
.
& C chix against C hen & C Chix & AA chix against C hen & C chix & AA chix against C hen & AA chix & AA chix against C hen & C chix
1 wk.
2 wks.
4 wk. M
+3.8 + 1.7 +4.0 +5.7
+2 +4 +5 +9
+8 +22 +2 +24
4 wk. F. -1
+1 +1 +2
1
449 chicks. Abbreviations used: AA=arsanilic acid, AB = antibiotic, AAAB = AA+AB, C = control diets, Chix = chickens, M = male, F = female. 2
creased and 1 weighed the same). When the progeny of dams fed arsanilic acid supplement were themselves fed .02 % arsanilic acid they increased body weight 11 out of 12 times over chicks fed the same diet but from dams without arsanilic acid treatment. Vitamin K deficiency symptoms. Observations in late September and early October (Experiment 2) revealed that chicks from the cage layers now entering their second year of lay were bleeding after wing banding. For the next 5 months all chicks from the cage groups were pedigree hatched and wing banded. Hemorrhages were scored the following day. Diets were calculated and found to contain the required 0.5 mg./kg. of menadione. Review of mixing procedure eliminated any reasonable chance that vita-
min K had been omitted from the premix. The vitamin premix in question was then approximately 4 months old but had been stored under refrigeration. As an initial check that a vitamin K deficiency existed, 12 hens with the highest number of afflicted chicks were given 2 intraperitoneal injections of 5 mgs. each of menadione sodium bisulfite in \ cc. of water. Injections were made on October 30 and November 9. Hemorrhages ceased immediately after these injections, while the controls continued with the same high incidence. It should be noted that at no time did the hens exhibit any evidence of improper diets and this vitamin deficiency would not have been noticed if the chicks that hatched had not been wing banded. This effect had been noted by Cravens et at. (1941).
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ARSANILIC ACID IN LAYING RATIONS 50 NEW PREMIX ADDED
. tt
MENADIONE SODIUM B I S U L F I T E
ADDED
^o •
/
\
S\.
30
20
9
10
*
V 1
10/11
10/25
1
-1
11/8
11/22
^ > 1
12/6
1
12/20
1
1/3
1/17
,. 1
1
\
V^N
1 —
*_l
1/31
2/14
2/28
3/7
WEEKLY HATCH DATES
FIG. 1. Incidence of wing hemorrhages induced by banding chicks hatched from vitamin K deficient dams.
Chick wing hemorrhages were diagnosed depending upon the degree of bleeding into 4 classifications: death (D), severe (S), intermediate (I), and small (s). During the early observations, an estimated onehalf of the hemorrhages were (S) or (I). This decreased to a small amount during the last hatches. A new vitamin premix (same formula, same menadione) was utilized during the last of October. Results of the new vitamin premix (Fig. 1) indicate an immediate reduction from 30% wing hemorrhages to as low as 10% wing hemorrhages among the chicks during the next 3 weeks. The incidence rose again to 25% bleeders and then slowly decreased to 9%. Menadione sodium bisulfite supplement (1.0 mg./kg., atomic weight 330) was substituted for menadione (atomic weight 172) and chicks from this combination were hatched in early February. By March 8, all chicks were normal. The 12% protein diets were not as severely affected as the 14 and 16% protein diets during the first 10-week period of observation (Table 4). During the last 8 weeks there was no difference in bleeding.
The arsanilic acid supplemental groups were detrimentally affected. At the end of the first 10-week summary the control groups were experiencing 12, 28, and 26% incidence of bleeding while the comparable arsanilic acid groups had 21, SO and 35% wing hemorrhage. The last 8-week summary (January 3 to March 7) shows the control group averages to be 2, 3 and 7% bleeders while the arsanilic acid groups were 12, 14, and 4% bleeders respectively. By March 7 all groups were fully recovered. Although these findings were obtained
TABLE 4.—Percentage of vitamin K deficient cage
hens' of spring suffering wing band bleeding Diet percent1 protein
Chick hatching dates Oct. 18, March 8, (experiment 2) Summary 10 hatches to 12/20
Summary 8 hatches to 3/7
12% 12%AA 12%AAAB
12 21 22
2 12 8
1,213/181 (14.9%)
14% 14%AA 14%AB 14%AAAB
28 50 31 32
3 14 8 12
1.406/362 (25.7%)
16% 16%AA
26 35
7 4
726/148 (20.4%)
1
No. eggs/bleeders
Abbreviations: AA=arsanilic acid 0.02%, AB =aureomycin 20 gms./ton, AAAB =both AA and AB.
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D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
with the breeding hen fed a diet supplemented with .02 % arsanilic acid, they substantiate the reports of Griminger et al. (1953) who noted a significant increase (P < .05) in blood clotting time in 4 week old chicks fed .01% arsanilic acid. Menadione (5 mgs./kg.) significantly reduced blood clotting time in the controls and among the arsanilic acid fed chicks. Griminger (1962) repeating the experiment again concluded that arsanilic acid modified the intestinal synthesis of vitamin K, but was not antagonistictoit. Chicks from antibiotic fed dams or antibiotic and arsanilic acid supplemented dams were intermediate in response between the arsanilic acid treatments and the controls. This was in agreement with Nelson and Norris (1959). Review of the individual hens with large
numbers of chicks having wing hemorrhages as well as hens having chicks with no wing hemorrhages reveals no relationship with the incidence of blood spots in eggs. While vitamin K was involved with the wing hemorrhages in the banded baby chicks it apparently was not related to the blood spot incidence in the eggs as the blood spot frequency (Table 5) was still increasing in January when the chick hemorrhages were greatly decreased. This observation would be in agreement with Perdue et al. (1961) who noted no relationship between vitamin K and blood spot incidence. Blood spot incidence. Nine observations were made of the cage birds' eggs starting with the initial pre-experimental period. Later, examination was made of samples of
TABLE 5.—Percent blood spot occurrence in cage layers fed arsanilic acid and antibiotic at three dietary protein levels Experimental periods (experiment 2)
Pre exp. Oct.
Dec.
Jan.
May
Oct.
Nov.
Dec.
Jan.
12% 12%AA 12%AAAB Average
0 2.4 0 0.8
0 0 0 0
0 3.3 0 1.0
0 0 0 0
4.2 4.8 0 3.0
8.0 0 3.8 4.0
5.0 7.3 5.0 5.8
3.7 3.6 0 2.7
14% 14%AA 14%AB 14%AAAB Average
0 4.6 2.5 0 1.8
10.0 10.5 0 0 4.9
0 12.8 2.6 2.7 4.6
0 14.3 0 0 3.9
0 0 0 0 0
0 3.6 7.7 0 2.7
6.3 15.6 3.3 4.8 7.5
2.5 23.8 0 0 4.2
16% 16%AA Average
5.1 4.4 4.8
0 5 2.4
0 13.9 6.8
5 8.3 6.6
0 16.7 7.7
4.0 33.3 16.3
8.5 27.0 15.9
19.2 31.6 24.4
Grand Ave.
2.1
3.0
4.0
3.4
2.6
5.7
8.7
7.6
ooo
Diet percent protein 1
0 0 4.0
6.8 12.6 3.4
4.0 11.8 9.1
Adjusted data 3
12%AA 14%AA4 16%6 1
0 0 2.8
0 6.7 0
3.7 12.9 0
0 11.4 2.7
2
Abbreviations used: AA=arsanilic acid, AB = antibiotic, AAAB = both AA and AB. Adjusted data corrected by removal of hens whose eggs contained blood spots during control period and more than 1 occurrence later. 3 Removed hen B 26 (29 eggs, 3 blood spots). 4 Removed hen F 15 and F 5 (47 eggs, 13 blood spots). 6 Removed hen H 102 (22 eggs, 10 blood spots). 2
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ARSANILIC ACID IN LAYING RATIONS TABLE 6.—Blood spot incidence in cage layers fed arsanilic acid and antibiotic at three protein levels {Adjusted data)1 Experiment 2 Percent protein diet2
Pre exp. diet Hens affected
Hens affected3
HensX breakout periods4
12% Control 12%AA 12%AAAB Total
-0-0-0-0-
3/12 3/12 4/12 10/36
6/96 5/96 5/96 16/288
14% Control 14%AA 14%AB 14%AAAB Total
-0-01/12 -01/48
5/12 7/12 3/12 5/12 20/48
9/96 14/96 N S 6/96 5/96 34/384
16% Control 16%AA Total
1/12 2/12 3/24
5/12 7/12 12/24
7/96 22/96*** 29/192
Eggs/Blood spot No. 223/8 214/7 232/5 669/20 281/9 223/21 275/6 246/5 1,025/41 245/7 210/41 455/48
% 3.59 3.27 2.15 2.98 .20 41* .18 ,03 .00 2.86 21.90*** 10.55
** Significant at .01 level.6 *** Significant at .005 level. Adjusted data corrected by removal of 4 birds whose eggs contained blood spots during pre-experiment period and more than 1 occurrence during experiment. 2 Abbreviations: AA=arsanilic acid, AB = antibiotic, AAAB = AA and AB. 3 Additional hens laying blood spots. Does not include blood spot layers during pre-experiment period. 4 Number of hens in the group laying one or more bloodspot eggs during each breakout period. 6 Determined by x 2 f ° r interaction and normal approximation (Steel and Torrie, 1960). 1
eggs laid by the sibs of these birds maintained in floor pens. Breakout periods were extended to as long as 20 days to reduce variation which occurred during the earlier 3-day breakout periods. All blood spots were recorded. These varied from pin point size to large masses although the latter were few. Standard breakout equipment was used and all eggs were at least 24 hours old. Summarization of the pre-experimental data (taken when the birds first started laying) by percent based upon the individual hens (Table 5) indicated that 8 of the 108 birds laid an egg containing 1 or more blood spots during the pre-test period of 5 days. Two of these hens laid no further blood spots, 2 laid 1 each. The remaining 4 hens were removed from the analysis. Table 6 excludes the 4 bloodspot prone birds. The column headed Hens Times Breakout Periods considers the persistency with which a hen laid blood spot eggs dur-
ing the 8 separate breakout periods. Observations based upon the control groups (Table 6) indicate that the persistency (6/96, 9/96, 7/96) and intensity (3.6%, 3.2%, and 2.9%) with which the hens continued to lay blood spots remained approximately the same at all protein levels. These data were found to be in disagreement with Bearse et al. (1962) who reported a positive association between blood spot incidence and increased dietary protein levels of 14, 16, and 18%. Upon the inclusion of .02% arsanilic acid the 14 and 16% protein diets appeared to increase the probability of a hen to lay one or more blood spots during her lifetime (12% diet 5/96, 14% diet 14/96, and 16% diet 22/96). The 16% protein diet supplemented with arsanilic acid was significantly greater (P < .005) than the control group in the numbers of hens and the persistence with which they continued to lay blood spot eggs. Among these cage layers, arsanilic acid,
1312
D. K. ANDREWS, H. R. BIRD AND M. L. SUNDE
while having no effect at the 12% protein level, produced significantly higher numbers of blood spots (P <.005) at the 14 and 16% protein levels as determined by adjusted x2. Omitting the 4 questionable hens changed the "/2 values only slightly, causing the 14% adjusted group comparison to drop to P <.01. Treatments containing 20 grams per ton of antibiotic alone and with arsanilic acid had the lowest blood spot incidence (2% and 2.2%) in the 12 and 14% protein groups. This beneficial response to chlortetracycline is contrary to the reports of other research workers. Chin and Brant (1953), Bearse and Berg (1955), and White-Stevens (1963) report no difference. A comparison of the U.S.D.A. Egg Production Tests summary data (1961, 1962) substantiates the results found at this station. Six of the Random Sample Test rations had an antibiotic supplement and 6 had no antibiotic. Blood spot incidence among the 6 without antibiotic supplement was. 4.9% the 1st year and 5.3% the 2nd year. Those with antibiotic supplement had 3.3 and 3.1% incidence of blood spots. Correspondence confirmed that no changes had occurred in the diets. Inspection of the 1963 and 1964 reports reveals that the same relationship continued. Daily blood spot incidence and frequency varied greatly, fluctuating from 0 to 10% and 0 to 33%, by periods (Table 5). Seasonal differences were noticeable and appear to have increased during the 2nd year of lay. A comparison of cage layers to their floor pen sisters on identical diets indicates substantially the same information as that found among the cage birds. Protein level was not associated with blood spot incidence. The 16% protein diets with arsanilic acid appeared to cause a significantly (P >.05) higher blood spot occurrence than was found in the control diet. Floor
pen control and adjusted battery controls had approximately the same percentages (12% diet 3.2-3.6; 14% diet 4.2-3.2; and 16% diet 2.2-2.9). Hens in floor pens fed arsanilic acid appeared to lay about the same number of blood spot eggs at all protein levels (12% diet 6.5%; 14% diet 4.8%; and 16% diet 5.8%). SUMMARY
Two years' observations of 12, 14, and 16% protein laying diets with and without .02% arsanilic acid in both cages and floor treatment show that: 1. Fertility and total number of chicks hatched were statistically improved by the addition of arsanilic acid. 2. The progeny from hens fed arsanilic acid exhibited slight growth increases over chicks from dams on corresponding control diets. This maternal effect was present over and above that found by feeding the chicks a .02% arsanilic acid supplemented diet. 3. Cage birds receiving 14 and 16% protein diets with .02% arsanilic acid increased significantly in blood spot incidence. To a lesser degree this was also true in the floor pens at all protein levels. 4. Hens fed .02% arsanilic acid diets had a higher minimal vitamin K. requirement than sibs on corresponding control diets. REFERENCES Andrews, D. K., H. R. Bird and M. L. Sunde, 1966. The effects of arsanilic acid on laying hens at three dietary protein levels. Part I. Egg production. Poultry Sci. 45: 838-847. Barnett, B. D., D. J. Richey and C. L. Morgan, 1959. The effect of feeding terramycin, furazolidone, and arsenicals to breeder hens. Assoc, of Southern Agr. Workers, Memphis, Tenn. p. 237. Bearse, G. E., and L. R. Berg, 1955. The effect of varying levels of aureomycin on the performance of young laying chickens. Poultry Sci. 34 : 1180. Bearse, G. E., L. R. Berg and A. H. Massey, 1962.
ARSANILIC ACID IN LAYING RATIONS Protein level of the diet and incidence of blood spots in chicken eggs. Poultry Sci. 4 1 : 1625. Carlson, C. W., 1957. Some effects of arsanilic acid and/or penicillin upon egg production. Poultry Sci. 36: 1070-1075. Carlson, C. W., 1959. Effects of antibacterial and antifungal agents in diets for laying and breeding hens. South Dakota Agr. Sta. Tech. Bui. No. 22: 1-23. Chin, G., and A. W. Brant, 1953. Egg quality and aureomycin. Poultry Sci. 32: 875-876. Cravens, W. W., S. B. Randle, C. A. Elvehjem and J. G. Halpin, 1941. Vitamin K studies 1. Effect of the vitamin K content of the hen's ration on the clotting ability of chick blood. Poultry Sci. 20: 313-316. Frost, D. V., 1953. Considerations on the safety of arsanilic acid for use in poultry feeds. Poultry Sci. 32: 217-225. Griminger, P., H. Fisher, W. D. Morrison, J. M. Snyder and H. M. Scott, 1953. Factors influencing blood clotting time in the chick. Science, 118: 379-380. Griminger, P., 1962. Arsanilic acid and blood coagulation. Poultry Sci. 4 1 : 982-985. Kurnick, A. A., R. J. Pforsich, M. W. Pasvogel, H. B. Hinds and L. W. Dewhurst, 1960. Effects of furazolidone and arsanilic acid on the reproductive performance of hens under conditions of varying temperatures. Proceedings Third National Symposium on Nitrofurans. p. 136-150. Libby, D. A., A. C. Groschke, R. J. Evans and
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S. L. Bandemer, 1953. Feeding of arsanilic acid to pullets. Michigan Agr. Exp. Sta. Quart. Bui. 35: 419-427. Lillie, R. J., J. R. Sizemore and C. A. Denton, 1957. Effect of an arsenical, fat, and antibiotic upon the reproductive performance of chickens. Poultry Sci. 36: 755-759. Nelson, T. S., and L. C. Norris, 1959. Factors affecting the vitamin K requirement of the chick. Poultry Sci. 38: 1094-1102. Perdue, H. S., J. A. Kolar and D. V. Frost, 1961. Non-correlation of vitamin K status and production of eggs with blood spots. Poultry Sci. 40:1443. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw-Hill Book Co., Inc., New York, N.Y. Thornton, P. A., and R. E. Moreng, 1958. Egg production and reproduction as affected by arsanilic acid and penicillin. Poultry Sci. 3 7 : 554557. United States Department of Agriculture, 1961. Report of egg production tests for the period 1960-1961. ARS 44-79-2. United States Department of Agriculture, 1962. Report of egg production tests for the period 1961-1962. ARS 44-79-3. Wharton, F. D., Jr., and J. C. Fritz, 1953. Studies on the effect of an arsenical in the diet of the immature and mature chicken. Poultry Sci. 32 : 1014-1020. White-Stevens, R. H., 1963. Personal Communication. American Cyanamid Co., Princeton, N.J.
Effect of Increasing Dietary Levels of Acetyl-Salicyhc Acid on Performance and Cecal Microbial Counts of White Leghorn Pullets1 J. M. THOMAS,2 H. S. NAKAUE AND B. L. REID Poultry Science Departent, University of Arizona, Tucson, Arizona (Received for publication April 7, 1966) INTRODUCTION
AN earlier report from this station, Reid A et al. (1964), showed that when 0.05% acetyl-salicylic acid (ASA) was fed 1
Arizona Agricutural Experiment Station Technical Paper No. 1111. 2 Present address: Batelle 'Memorial Institute, Richland, Washington.
White Leghorn pullets either alone or in combination with 27.5 mg. oxytetracycline per kg., a significant increase in egg production resulted. The improvement was independent of periods of high ambient temperature and thus the action of the drug was assumed to be exclusive of its antipyretic properties.