Whole Blood Ascorbic Acid Levels in Chickens with Experimental Aplastic Anemia and the Effect of Supplemental Ascorbic Acid, B12, and Minerals on Mortality and Pathologic Manifestations

Whole Blood Ascorbic Acid Levels in Chickens with Experimental Aplastic Anemia and the Effect of Supplemental Ascorbic Acid, B12, and Minerals on Mortality and Pathologic Manifestations

Whole Blood Ascorbic Acid Levels in Chickens with Experimental Aplastic Anemia and the Effect of Supplemental Ascorbic Acid, B12, and Minerals on Mort...

326KB Sizes 0 Downloads 26 Views

Whole Blood Ascorbic Acid Levels in Chickens with Experimental Aplastic Anemia and the Effect of Supplemental Ascorbic Acid, B12, and Minerals on Mortality and Pathologic Manifestations P . D . MOORHEAD AND R. F . CROSS Department of Veterinary Science, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691 (Received for publication February 9, 1970)

HE hemorrhagic and anemic manifestations of aplastic anemia in chickens resulting from sulfaquinoxaline toxicity described by Sadek etal. (1955) and Sanger et al. (1956) are similar to lesions reported for scurvy in man, guinea pig and monkey by Duncan (1947) and Jolliff et al. (1950). Roy and Guha (1958) have reported extensive hemorrhage as a manifestation of experimental scurvy in a species of bird known to require a dietary source of ascorbic acid. Although clinical scurvy in chickens is not known to occur, some recent studies have indicated that the ascorbic acid synthesizing mechanism in the chicken may be inadequate or defective during periods of stress. Ahmad et al. (1967) have shown that dietary ascorbic acid supplementation was of value in counteracting high environmental temperature conditions for laying birds. Challey (1960) reported a marked increase in adrenal ascorbic acid in chickens subjected to experimental hemorrhage and intestinal coccidiosis. Results from these studies suggest that chickens stressed for a prolonged period may be unable to synthesize sufficient ascorbic acid to prevent clinical deficiency. Chatterjee et al. (1961) reported the principal site of ascorbic acid synthesis in the chicken was in the kidney microsomes. The kidney is also the organ most severely affected by sulfaquinoxaline toxicity in the chicken (Garner, 1963). The hemorrhagic and anemic manifestations of sulfaquinox-

aline toxicity in the chicken may be the result of interference with synthesis of ascorbic acid by the kidney and/or an alteration in its metabolic function in erythropoietic centers. The present study was designed to provide basic data on whole blood levels of ascorbic acid in chickens with sulfaquinoxaline induced aplastic anemia and the effect of exogenous ascorbic acid, copper, iron, and B12 on the hemorrhagic and anemic manifestations of the syndrome. MATERIALS AND METHODS

Sixty-three one-day-old commercial broiler chicks were divided into three groups of twenty-one each and reared for six weeks in a brooder battery. Each group was placed in an isolation room for the remaining four weeks of the experiment. The temperature in the isolation rooms was maintained at a constant 28°C. (82°F.). Sulfaquinoxaline was mixed in the ration at a level of 0.15% and fed to the chicks in each group for a period of 28 days (Garner, 1963). The ingredients of the basal ration (BR) and vitamin-premix are shown in Tables 1 and 2. The ingredients and levels of the experimental supplement (ES) are shown in Table 3. The basal ration was used as a carrier for sulfaquinoxaline (SQ) and the experimental supplement. The rations were compounded and mixed 1 week prior to the initiation of the trials. The levels of copper, iron, and B12 used in the ex-

1065

Downloaded from http://ps.oxfordjournals.org/ by guest on May 26, 2015

T

1066

P . D . MOORHEAD AND R . F . CROSS

TABLE 1.—Composition of basal ration used for experimental aplastic anemia in chickens Composition Percent

Pounds

34.7 39.0

695 780

5.0 5.0

100 100

2.5

50

2.5

50

5.0 0.9 1.2 0.4 1.5 2.25

100 18 24 8 30 45

Group

Diet, Diet, 1 through 6 weeks 7 through 10 weeks BR BR+ES BR

BR+ES+SQ BR+ES+SQ

BR+SQ

perimental supplement were reported by Ewing (1963) and Washburn and Huston (1968) to be adequate when used to correct known deficient diets. Ascorbic acid supplementation was based on the level reported by Ahmad et al. (1967) to be effective in counteracting stress produced by high environmental temperatures on laying birds. Ascorbic acid assay of the supplemented rations at initiation of the feeding trials was 4.1 mg. per 100 gms. feed and at the end of the 10-week trial period the level was reduced to 0.95 mg. ascorbic acid per 100 gms feed. Assay of nonsupplemented basal ration revealed no significant level of ascorbic acid. Ascorbic acid levels in the ra-

Heparinized blood samples were collected from each bird prior to the administration of sulfaquinoxaline and at 2-week intervals thereafter. Packed cell volume (PCV) and whole blood ascorbic acid level determinations were conducted on each sample within 1 hour after collection. Total ascorbic acid determinations were conducted on 2 ml whole blood samples using the methods described by Shaffert and Kingsley (1955). Necropsy examinations were made on all dead of moribund birds. The remaining live birds were killed for necropsy examination at the end of the 10th week. The following organs and tissues of all birds were examined for gross lesions: skeletal muscle, subcutis, thymus, heart, liver, spleen, kidneys, small intestine and marrow of proximal end of the femur.

TABLE 2.—Composition of premix V-3-68 used in basal ration for experimental aplastic anemia in chickens

TABLE 3.—Composition of experimental supplement tested for effect on experimental aplastic anemia in chickens

Supplement Vitamin A Vitamin Da Vitamin E Riboflavin Pantothenic acid Niacin Methionine Antioxidant (BHT) Soybean meal (44% protein) Choline chloride Penicillin Ground yellow corn

Potency of Amount for supplement 1 ton 20,000 units/gm. 200 gms. 3,000 units/gm. 540 gms. 182 gms. 20,000 units/454 gms. 120 gms. 35 mgs./454 gms. 70 mgs./gm. (Ca salt) 70 gms. 1,000 mgs./gm. 50 gms. 908 gms. 900 mgs./gm. 454 gms. 250 mgs./gm. 476 gms. 25 gm./lOO gms.

2000 gms. 4 gms. 15.45 kg.

Supplement

Copper Iron Cyanocobalamin Ascorbic acid

Form

Amount added to 10 lbs. (45.36 kg.) of ration

CuS0 4 -5H 2 0 Ferric ammon. citrate Pure

3.56 gms. 27.51 gms.

Reagent grade

2.0 gms.

0.8

mg.

Downloaded from http://ps.oxfordjournals.org/ by guest on May 26, 2015

Ground yellow corn Soybean meal (44% protein) Wheat middlings Meat and bone scrap (50% protein) Menhaden fish meal (60% protein) Dried whey product (16% protein) Alfalfa meal (17% protein) Biofos Ground limestone Trace mineral poultry salt Dried fish solubles Premix V-3-68

tions were estimated by the methods described by Shaffert and Kingsley (1955). The experimental supplement was designed to provide adequate levels of the added nutrients without regard to the levels existing in the basal ration. The diet regimen for each of the three groups of birds was as follows:

1067

APLASTIC ANEMIA TABLE 4.—Packed cell volume and blood ascorbic acid levels of chickens that died of sidfaquinoxaline induced aplastic anemia

Group

Period* Mortality supplement • fed, in No. % wks. group total

6 to 10

1 ro 10

5

4

6

23.8

20

28.6

Blood Ascorbic Acid mg./lOO ml.

Packed Cell Volume Pre-sulfa

2 weeks post-sulfa

4 weeks post-sulfa

Pre-sulfa

2 weeks post-sulfa

4 weeks post-sulfa

mean range

mean range

mean range

mean range

mean range

mean range



0.98 to 1.10 1.21

0.98 to 1.17 1.45

1.29

0

0

0.97 0.98 to to 1.04 1.17 • ,1.04 1.10

0

0

0

0.91 to 1.01 "1.29

0

0

26.4 24-29

29.2 27-31

28.1 24-32

22.0 14-26

15.6 5-22

26.2 22-29

28 b

0.98 to 1.18 1.58

RESULTS

The effect of the experimental supplement on the mortality rate of birds in groups 1 and 2 was not significant when compared with group 3 controls (Table 4). One chick in group 2 died prior to the initiation of feeding the experimental ration. Total ascorbic acid levels were not significantly altered in whole blood of any of the three groups (Tables 4 and 5). The levels in most of the birds tested fell close to the normal range of 1.07 mg. to 1.62 mg. per 100 ml. whole blood reported by Simmonds (1965). Least squares analysis of variance of the data in Tables 4 and 5 revealed no significant correlation of whole blood levels of ascorbic acid with PCV values in any of the 3 groups. The PCV values declined (Table 5) in birds in all three groups that survived the 10-week experimental period. It is of interest that group 1 exhibited the least variation in PCV, and was correlated with the absence of hemorrhage and a low percentage of bone marrow hypoplasia. Hemorrhage and bone marrow hypoplasia exhibited by birds in group 2 were slightly less frequent than those found in control group 3 (Table S). Two birds in each group died of conditions unrelated to

sulfaquinoxaline toxicity and were not included in the tabulated data. DISCUSSION The whole blood levels of ascorbic acid in chickens with sulfaquinoxaline induced aplastic anemia suggests that the mechanism of toxicity was neither depletion of ascorbic acid nor inhibition of its synthesis. Hemorrhagic manifestations of scurvy in man and guinea pig discussed by Jolliff et al. (1950) seldom appeared prior to zero or near zero plasma levels of ascorbic acid. It has been observed by Duncan (1947) in cases of human scurvy that oral administration of ascorbic acid was much less effective than parenteral therapy in alleviating hemorrhagic manifestations and anemia. The excessive expense and labor involved would not justify the use of parenteral ascorbic acid therapy for aplastic anemia in chickens without substantial proof of efficacy. The absence of hemorrhage and the low percentage of bone marrow hypoplasia found in birds from group 1 was not considered totally the result of feeding the experimental supplement as the birds in group 2 received the same ration for a much longer period. A review of manage-

Downloaded from http://ps.oxfordjournals.org/ by guest on May 26, 2015

* Ascorbic acid, Fe, Cu, and B12. Moribund on day of final blood sample collection. Highest mortality occurred between the 14th and 28th day and only one bird lived to fourth week test period.

b 0

1068

P . D . MOORHEAD AND R . F . CROSS

-H

TH

O

-H O

-H

*ik




O •*



-HO

• -4-»

.

-H O

• *->

W

en

o

00 OCN00 O ^ O N o tO

3 «

O

-HO

.-< O

-H

"SI

a.« 0\

|1

I

CJ

O to I

e I

^1 7~

•* •<*'

•^ 2

PM

is

SJS

6§£

3

s.s

II


Ills* PH

en

§• O

O

o o

3-

II

II

SUMMARY Whole blood level ascorbic acid determi-

Downloaded from http://ps.oxfordjournals.org/ by guest on May 26, 2015

CM

ment practices, room temperatures and rations revealed no obvious explanation for the difference in frequency of lesions occurring in birds in groups 1 and 2. The rapid degradation of L-ascorbic acid when mixed in the basic ration was demonstrated by only 23.1% of the original vitamin activity remaining after the 10-week period. Estimating the average feed consumption, per bird at 10 weeks old, to be SO gms. per day, the ascorbic acid intake would be approximately 0.62 mg. per kg. daily with an 0.95 mg. per 100 gm. level in the ration. The estimated minimum ascorbic acid intake required by an adult human for prevention of clinical scurvy suggested by Jolliff et al. (1950) is 15 mg. daily or hypothetically 0.33 mg. per kg. of body weight for a 45.45 kg. adult. On this basis, there should have been sufficient ascorbic acid remaining in the experimental ration to have been effective had exogenous ascorbic acid been a factor in maintaining normal blood levels in the birds. Had exogenous ascorbic acid been a modifying factor, loss of its activity from the ration and decreased feed intake by intoxicated birds should have been reflected by decreased blood levels as the birds' tissue levels were depleted. As there appeared to be no significant difference in whole blood levels of ascorbic acid from birds receiving an exogenous source and the control birds, it was concluded that supplemental ascorbic acid in the ration was of no value in preventing sulfaquinoxaline intoxication in the chicken. In conclusion, supplementation of rations with ascorbic acid, copper, iron, and B12 with conditions similar to the experimental trials would be of little significant value in decreasing the mortality of chickens fed toxic levels of sulfaquinoxaline.

APLASTIC ANEMIA

nations were made from chickens with sulfaquinoxaline induced aplastic anemia. The total ascorbic acid blood levels in the experimental birds were not significantly altered by either sulfonamide intoxication or ascorbic acid supplementation of the diet. No significant correlation of whole blood levels of ascorbic acid with packed cell volume levels was observed in any of the experimental groups. Supplementation of the diets with ascorbic acid, copper, iron, and B12 had no notable effect on the mortality rate of birds in the treated groups.

Ewing, W. R., 1963. Poultry Nutrition, Fifth Edition, Ray Ewing Co., Publisher, 2690 Foothill Blvd., Pasadena, Calif, p. 868-887. Garner, R. J., 1963. Veterinary Toxicology, Second Edition, Williams and Wilkins Co., Baltimore, O. p. 201-202. Jofliff, E. E., F. F. Tisdall and P. R. Cannon, 1950. Clinical Nutrition, Paul B. Hoeber Inc., Harper and Brothers, New York, N.Y. p. 587601. Roy, R. N., and B. C. Guha, 1958. Production of experimental scurvy in a bird species. Nature, 182: 1689-1690. Sadek, S. E., L. E. Hanson and J. O. Alberts, 1955. Suspected drug induced anemias in the chicken. J. Amer. Vet. Med. Assoc. 127: 201203. Sanger, V. L., H. Yacowitz and E. N. Moore, 1956. Micropathological changes in experimental hemorrhagic syndrome in chickens fed sulfaquinoxaline and suggested cause of the disease. Am. J. Vet. Res. 17: 766-770. Shaffert, R. R., and G. R. Kingsley, 1955. A rapid simple method for the determination of reduced dehydro- and total ascorbic add in biological material. J. Biol. Chem. 212: 59-68. Simmonds, R. A., 1965. Ascorbic acid levels in the blood of growing chickens. Poultry Sci. 44: 308-310. Washburn, K. W., and T. M. Huston, 1968, Effects of environmental temperatures on iron deficiency anemia in Athens-Canadian random bred chicks. Poultry Sci. 47: 1532-1535.

Effect of Method of Determination on the Metabohzable Energy Value of Rapeseed Meal1 P. V. RAO2 AND D. R. CLANDININ Department of Animal Science, The University of Alberta, Edmonton, Alberta, Canada (Received for publication February 9. 1970)

ALTHOUGH the use of rapeseed meal -*V. (RSM) in poultry rations has been extensively studied (reviewed by Clandinin and Robblee, 1966), reports on the metab1 Supported in part by grants from the National Research Council of Canada and the Canada Department of Agriculture. 2 Postdoctoral Fellow at The University of Alberta.

olizable energy (ME) value of RSM are few in number. Sibbald and Slinger (1963b) reported a value of 1670 kcal./kg. for one sample of RSM with chickens and Sell (1966) obtained a value of 2290 kcal./ kg. for one sample of RSM with hens. Recently, Lodhi et al. (1969b), employing the ME method of Hill and Anderson (1958), obtained average ME values for nine sam-

Downloaded from http://ps.oxfordjournals.org/ by guest on May 26, 2015

REFERENCES Ahmad, M. M., R. E. Moreng and H. D. Muller, 1967. Breed response in body temperature to elevated environmental temperature and ascorbic acid. Poultry Sci. 46: 6-15. Challey, J. R., 1960. The effect of cecal coccidiosis infections and experimental hemorrhage upon adrenal ascorbic acid levels in the chicken. J. Parasit. 46: 727-731. Chatterjee, I. B., N. C. Carr, N. C. Ghosh and B. C. Guha, 1961. Aspects of ascorbic acid biosynthesis in animals. Ann. New York Acad. Sci. 92: 36-56. Duncan, G. G., 1947. Diseases of Metabolism, Second Edition, W. B. Saunders Co., Philadelphia, Pa. p. 366-367.

1069