Chick Bioassay of Vitamin K Compounds Using Dicumarol and Pivalyl as Anticoagulants 1

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1 754 F. L. CHERMS Kosin, I. L., Metabolism of turkey semen as affected by the environment of donor birds. Poultry Sci. 37: Lake, P. E., Artificial insemination in poultry. Adv. Repr. Physiol., Vol. I. Logos Press Ltd., London. Law, G. R. J., and I. L. Kosin, Seasonal reproductive ability of male domestic turkeys as observed under two ambient temperatures. Poultry Sci. 37 : Lorenz, F. W., Reproduction in the domestic fowl. In Reproduction in Domestic Animals, Vol. 2, Academic Press, Inc., New York. McCartney, M. G., Relation between semen quality and fertilizing ability of White Holland turkeys. Poultry Sci. 35: McCartney, M. G V. D. Chamberlin, R. D. Carter and J. W. Wyne, Effect of frequency of semen collection on fertility, hatchability and CHICK bioassay procedures have been widely used to study the relative efficacy of vitamin K compounds (Perdue and Frost, 1960; Griminger, 1965; Dua and Day, 1966). Since the reduction of blood prothrombin time is the criterion used in such studies, a stress agent to sensitize the assay is desirable. Despite certain adverse side effects, sulfaquinoxaline has been used as the anticoagulant stress agent in most previous work. Snake venom has been used as the sensitizing agent on a limited basis (Matschiner and Doisy, 1966). Toxic effects of sulfaquinoxaline have been studied by Delaplane and Milliff (1948), Newbern and Buck (1956), Sanger et al. (1956) and others. Sanger et al. (1956) found that experimental birds receiving 0.1 percent sulfaquinoxaline in spermatozoa concentration in turkeys. Poultry Sci. 37: Parker, J. E., Semen production in Broad Breasted Bronze turkeys. Poultry Sci. 25: Saeki, Y., and K. I. Brown, Effect of abnormal spermatozoa on fertility and hatchability in the turkey. Poultry Sci. 41: Snedecor, G. W., Statistical Methods. 4th Ed. The Iowa State College Press, Ames, Iowa. Verma, O. P., and F. L. Cherms, The appearance of sperm and their persistency in storage tubules of turkey hens after a single insemination. Poultry Sci. 44: White, I. G., and G. J. Lincoln, The yellow pigmentation of bull semen and its content of riboflavin, niacin, thiamine, and related compounds. Biochem. J. 76 : Chick Bioassay of Compounds Using and as Anticoagulants 1 O. W. CHARLES, BEN C. DILWOETH AND ELBERT J. DAY Department of Poultry Science, Mississippi State University, State College, Mississippi (Received for publication September 8, 1967) the feed along with alfalfa meal and vitamin K active compounds developed hemorrhages, leukocytic infiltrations, bone marrow hypoplasia and anemia. Yacowitz et al. (1955) were unable to completely protect the chicks against the toxic effects of sulfaquinoxaline fed at the 0.1% level. Cuckler and Ott (1955) reported that diets containing alfalfa meal and less than 0.4% sulfaquinoxaline did not cause a prolonged prothrombin time. The reported ratio of activity of certain vitamin K active compounds has varied greatly in different investigations. For example, Frost et al. (1956) reported menadione sodium bisulfite complex () 2 to be three times as effective as menadione, while Shelton et al. (1956), reported an even greater ratio of activity. Perdue et 1 [Mississippi Agricultural Experiment Station Journal Article number Menadione sodium bisulfite complex contains 32.8% combined Menadione.

2 BIOSSAY OF VITAMIN K COMPOUNDS 755 al. (1957) found 1.5 times as effective as vitamin K x in the modified ANRC rations, while Nelson and Norris (1958) using a purified ration, reported vitamin Ki and to be equally active; however, both were found to be twice as effective as menadione. Griminger and Donis (1960) compared the relative efficacy of vitamin Ki with menadione and a bisulfite adduct. When using a diet low in vitamin K activity, supplemented with sulfaquinoxaline, these workers reported vitamin Ki to be 1.5 times as active on an equimolar basis as and 2.5 times as active as menadione. The purpose of these studies was to compare the ratios of activity of two water soluble vitamin K active compounds when using varying levels of either dicumarol or pivalyl as an anticoagulant stress agent. EXPERIMENTAL Four experiments were conducted with day-old broiler type chicks obtained from a commercial hatchery. Male chicks were used in Experiments 1 and 2, while chicks of both sexes were used in experiments 3 and 4. The basal diet was a corn-soybean meal type ration designed to be deficient in vitamin K (Table 1). All chicks in each experiment were fed the basal diet for 1 week. At one week of age, all chicks were weighed and 10 chicks of approximately equal weight (± 10 grams) were randomly assigned to each of 16 groups in each experiment. The experimental diets were formulated by supplementing the basal diet with the anticoagulants dicumarol or pivalyl and were fed with graded levels of or. 3 Both and were compared on an equal weight basis at four dif- 3 Menadione dimethylpyrimidinol bisulfite, a patented product of Heterochemical Corp., Valley Stream, New York, contains 45.5% combined menadione. TABLE 1. Composition of the basal diet Ingredients Percent Yellow corn Soybean meal (50% protein) Vegetable oil 3.00 Defl. rock phosphate 1.85 Ground limestone 0.25 Mineral vit. mix Total Calculated analysis: Crude protein, % Energy, prod. Cal./lb Calorie/protein 44.9 Phosphorus, % 0.64 Calcium, % 0.83 Methionine, %/l, 000 Cal Supplies the following per kg. of diet: vitamin A, 7,500 LIT.; vitamin D 3, 2,010 I.C.U.; vitamin E, 2.2 I.U.; vitamin B12, 0.01 mg.; riboflavin, 4.4 mg.; Ca-pantothenate, 8.8 mg.; choline chloride, 375 mg.; methionine hydroxy analogue, 0.45 gm.; salt, 3.8 gm.; manganese, 60 mg.; iodine, 1 mg.; iron, 16 mg.; copper, 2 mg.; zinc, mg. and cobalt, 0.4 mg. ferent levels each (.055, 0, 0 and 0 mg./kg. of diet). The birds were maintained in electrically heated battery brooders on raised wire floors. Feed and water were supplied ad libitum. The experimental diets were fed up to 3 weeks of age when all chicks were weighed individually and 4 chicks per dietary treatment were used to determine the average prothrombin time. Plasma prothrombin time was determined by a method similar to that of Quick and Stefanini (1948). The data were analyzed by the analysis of variance technique of Steel and Torrie (1960). The relative ratio of vitamin K activity of and was determined using the parallel line bioassay procedure as described by Davies (1958). RESULTS AND DISCUSSION The results of Experiment 1 are shown in Table 2. fed at the rate of 2.2 mg./kg. of diet was significantly more effective in increasing plasma prothrombin time (P < 0.01) than dicumarol fed at the rate of 44 mg./kg. of diet. This finding

3 756 O. W. CHARLES, B. C. DILWOETH AND E. J. DAY TABLE 2. The prothrombin times of male broiler chicks treated with pivalyl and dicumarol and receiving varying dietary levels of and (Exp. 1) Dietary level of vitamin K> (mg./kg..06 (44 mg./kg.) Av. 22 source Composit e data 2 66 A 51 B (2.2 mg./kg.) Pro thromb n Pro- thrombin Anticoagulant 21 A 96 B All chicks received the basal ration for the first week. From 1-3 weeks of age one half of the chicks received dicumarol (44 mg./kg.) and one half received pivalyl (2.2 mg./kg.) as stress agents. All birds received supplemental vitamin K activity as indicated. different at the 1% level of probability. agrees with a previous report (Charles et al., 1966) in which these two anticoagulants were studied and pivalyl was found to be much more potent than dicumarol. K H g 2.0 w g S 2 CO ll Si o Y X Y X o Y= X Y= X o B Griminger (1965) and Dua and Day (1966) reported a ratio of activity for and of about 1:2, respectively, using 0.1% sulfaquinoxaline as a stress agent in the ration. In the present experiment, with pivalyl as the anticoagulant, the ratio of activity of and was found to be 1:2, respectively, on an equal weight basis (Fig. 1A)., at the 44 mg./kg. of diet level, did not appreciably affect the prothrombin time in this experiment. No significant differences were found for rate of gain, feed conversion or mortality in Experiment 1. The results from Experiment 2 demonstrated the effect of higher levels of pivalyl on the ratio of activity of and. Highly significant differences were found both for vitamin K source and for anticoagulant level (Table 3). A ratio of activity of 1:1.9 for and, respectively, was calculated in the group receiving 11 mg. of pivalyl per kilogram of Y= X Y X ^ LOG VII. K, MCG/KG o Y= X Y X o Y= X Y= X A 2.2 mg/kg - Ratio 1:2 Exp. 1 B 11 mg/kg - Ratio 1:1.9 Exp. 2. C 11 mg/kg - Ratio 1:1.97 Exp. 4- D 2 mg/kg Males - Ratio 1:2.54 Exp.3 E 2 mg/kg Females - Ratio 1:10.4- Exp. 3 FIG. 1. Relationship of the log of the prothrombin time in seconds to the log of the meg. of or per kg. of diet.

4 BIOSSAY OF VITAMIN K COMPOUNDS 757 diet, Fig. IB. Chicks receiving 22 mg. of pivalyl per kg. of diet did not respond to graded levels of with a reduced prothrombin time (Table 4). Therefore, at this level of pivalyl it was not possible to calculate a ratio of activity for the two sources of vitamin K. In Experiment 3 chicks of both sexes were fed dicumarol (2 mg./kg.) as a sensitizing agent. was significantly more effective (P < 0.01) than in reducing the plasma prothrombin time (Table 5). No significant differences in prothrombin times were found between the three groups fed the lower levels of vitamin K, while the highest level ( mg./kg.) resulted in a significantly lower prothrombin time (P < 0.05) as compared to the other groups. Highly significant differences were found between sexes; however, sex differences occurred only in the treated birds and no significant difference was found between sexes for birds receiving. This sex response difference is possibly due to the fact that provided protection above the chick's requirement. It is noteworthy that chicks of both sexes TABLE 3. The effect of pivalyl level on the relative efficacy of and {Exp. 2), 11 mg./kg., 22 mg./kg. Dietary level of Pro- Pro- Pro- Provitamin thrombin thrombin thrombin thrombin K 1 (mg./kg.) Av Composite data 2 Anticoagulant source level level 136 A 22 mg./kg. 159 A.055 mg./kg. 139 a mg./kg. 126 ab 104 E 11 mg./kg. 82 E mg./kg. 115 bc mg./kg All birds were fed the basal ration for the first week of age, afterwhich, they were randomized and fed the experimental diets for a 2 week period. different at either the 5% level of probability (vitamin K source and anticoagulant level) or the 1% level (vitamin K level). TABLE 4. The effect of increasing doses of constant dose ratios of two vitamin K analogues and pivalyl on the plasma prothrombin time of chicks (Exp. 2) Ratio Levels (mg./kg.) Vit. K time receiving the highest level of exhibited a prothrombin time below the level of the untreated controls. The ratio of activity in this experiment for and was found to be 1:2.54 for males and 1:10.4 for females, respectively (Fig. ID and IE). (11 mg./kg. of diet) was used as the anticoagulant in Experiment 4. The prothrombin time was significantly lower in the groups supplemented with than that of those fed (Table 6). The TABLE 5. The ability of and to counteract the hypoprothrombinemia produced by dicumarol {Exp. 3) Male 2 Female 2 Vitamin K 1 Pro- Pro- Pro- Pro- (mg./kg.) thrombin thrombin thrombin thrombin.055 sources Av A 84 B Composite data 1 level.055 mg./kg. mg./kg. mg./kg. mg./kg. 121 a 126" 105 a 80 b Femal e Male Sexes A 1 All chicks were fed the basal ration for the first week. From 1-3 weeks of age all diets contained 2 mg. of dicumarol per kg. of diet and supplemental vitamin K as indicated. 2 time of untreated controls receiving only the low vitamin K basal ration averaged 49 and 69 seconds for males and females, respectively. 3 Drug means without a common superscript are significantly different at either the 1% level of probability (vitamin K source and sexes) or the 5% level (vitamin K level). 98 B

5 758 O. W. CHARLES, B. C. DILWORTH AND E. J. DAY TABLE 6. The prothrombin time of male and female broiler chicks treated with pivalyl and receiving varying dietary levels of and {Exp. 4) Vitamin (mg.ag-).055 sources Av A 82 B Male < Composite data 2 levels.055 mg./kg. mg./kg. mg./kg. mg./kg Female time. Sexes * Fen.ale 98* 101 B 80 Male B 66 D 1 All chicks were fed the basal ration for the first week. From 1-3 weeks of age all diets contained 11 mg. of pivalyl per kg. and supplemental vitamin K as indicated. different at the 1% level of probability. prothrombin time of female chicks was significantly greater (P < 0.01) than that of male chicks. The three highest levels of vitamin K caused highly significant (P < 0.01) differences in the prothrombin time. This is in contrast to the data obtained in Experiment 3 in which only the highest level of vitamin K activity elicited a significant prothrombin response. The prothrombin time of female birds receiving was significantly greater (P < 0.05) than that of the males. The ratio of activity for and was found to be 1:1.97, respectively (Fig. 1C). Since experiments 3 and 4 were conducted simultaneously using chicks from the same hatch and they were housed and raised in the same environments, these data were recombined for additional study (Table 7). The combined data show that the prothrombin time response to the two sources of vitamin K were highly significantly different. However, no significant difference was found at these levels between the anticoagulants. All significant interactions found in this analysis were due to source or level of vitamin K, not to the anticoagulant. In these experiments a level of 11 mg. of pivalyl/kg. of diet was equivalent to a level of 2 mg. of dicumarol in prolonging the plasma prothrombin time of the chick, indicating that pivalyl is times more potent than dicumarol. The ratio of biological activity between and was 1:2.54 for males and 1:10.4 for females, respectively, using dicumarol in Experiment 3. In Experiments 1 and 2 and 4, using pivalyl at the 2.2 and 11 mg./kg. level, respectively, ratios of 1:2 and 1:1. and 1:1.97 were obtained for and, respectively. These data suggest a basic difference in the mode of vitamin K antagonism of dicumarol and pivalyl. Griminger and Donis (1960) observed a differential response when studying different vitamin K sources with chicks treated with sulfaquinoxaline and dicumarol. The nature of the antagonism between vitamin Ki and warfarin has been studied by Lowenthall and McFarlane (1963). These workers suggested that in rats indirect anticoagulants antagonize the function of vitamin K at a specific site, and that, in TABLE 7. The ability of two anticoagulants to increase the prothrombin time of chicks receiving graded levels of and (Exps. 3 and 4) Dietary Plasma prothrombin Av source Composite data 2 Anticoagulant MBP 79^ 98^ 108 B A 1 All chicks were fed the basal ration for the first week. From 1-3 weeks of age all diets contained either 11 mg. of pivalyl P er kg. or 2 mg. of dicumarol and supplemental vitamin K as indicated. different at the 1% level of probability.

6 BIOSSAY OF VITAMIN K COMPOUNDS 759 addition to its normal site or mode of action, large doses of vitamin K x can act by an alternate site or mechanism. Their conclusions were based on observations of the initial increase of plasma clotting factors after the intravenous administration of vitamin KL Using water soluble vitamin K active compounds and a long term feeding trial, our results tend to agree with Lowenthall and McFarlane in that a constant dose ratio of antagonist did not cause a constant response (Table 4). Under the conditions of this experiment, our findings suggest that the ratio of the effective concentration changes with increasing doses. Sulfaquinoxaline, dicumarol, pivalyl and perhaps many other stress agents may be used effectively in studying the relative efficacy of vitamin K compounds. However, it is apparent from our data that the ratio of efficacy of vitamin K compounds are related to both the sex and the anticoagulant. SUMMARY and pivalyl were compared as anticoagulants in vitamin K studies. The relative efficacy of two water soluble vitamin K analogues, and, was determined using each of these anticoagulants as stress agents. Eleven milligrams of pivalyl per kg. of diet was equal to 2 milligrams of dicumarol per kg. of diet in prolonging the plasma prothrombin time of chicks. The relative efficacy for and was found to be 1:2.54 for males and 1:10.4 for females, respectively, when using dicumarol, while three similar tests using pivalyl at the 2.2 and 11 mg./kg. of diet yielded a 1:1.99 average ratio of activity for and, respectively. REFERENCES Charles, O. W., B. C. Dilworth, R. D. Bushong, Jr. and E. J. Day, The effect of dicumarol, diphacinone, and pivalyl upon blood prothrombin time of chicks. Poultry Sci. 45: Cuckler, A. C, and W. H. Ott, Tolerance studies on sulfaquinoxaline in poultry. Poultry Sci. 34: Davies, O. L., Statistical Methods in Research and Production. Hafner Publishing Company, New York. Delaplane, J. P., and J. H. Milliff, The gross and micropathology of sulfaquinoxaline poisoning in chickens. Am. J. Vet. Res. 9: -96. Dua, P. N., and E. J. Day, activity of menadione dimethylpyrimidinol bisulfite in chicks. Poultry Sci. 45: Frost, D. V., H. S. Perdue and H. C. Spruth, activity of menadione sodium bisulfite in chickens. J. Nutrition, 59: Griminger, P., and O. Donis, Potency of vitamin Ki and two analogues in counteracting the effects of dicumarol and sulfaquinoxaline in the chick. J. Nutrition, 70: Griminger, P., Relative vitamin K potency of two water-soluble menadione analogues. Poultry Sci. 44: Lowenthal, J., and J. A. McFarlane, The nature of the antagonism between vitamin K and indirect anticoagulants. J. Pharmacol. Exptl. Therap. 143: Matschiner, J. T., and E. A. Doisy, Jr., Bioassay of vitamin K in chicks. J. Nutrition, 90: Nelson, T. S., and L. C. Norris, Further studies on the chick's requirement for vitamin K,. Poultry Sci. 37: Newberne, P. M., and W. B. Buck, Studies on drug toxicity in chicks. 2. The influence of various levels of sulfaquinoxaline on growth and development of chicks. Poultry Sci. 35: Perdue, H. S., H. C. Spruth and D. V. Frost, Comparison of vitany'n K activities of vitamin Ki and Klotogen (Menadione sodium bisulfite complex). Poultry Sci. 36: Perdue, H. S., and D. V. Frost, Comparison of vitamin Ki and menadione sodium bisulfite complex as standards in chick diets suggested for vitamin K assay. Feedstuffs, 32: 34. Quick, A. J., and M. Stefanini, Experimentally induced changes in the prothrombin level of the blood. IV. The relation of vitamin K deficiency to the intensity of dicumarol action and to the effect of excess vitamin A intake; with a simplified method for vitamin K assay. J. Biol. Chem. 175: Sanger, V. L., H. Yacowitz and E. N. Moore, Micropathological changes in an experimental hemorrhagic syndrome in chickens fed

7 W. CHARLES, B. C. DILWORTH AND E. J. DAY sulfaquinoxaline and suggested cause of the disease. Am. J. Vet. Res. 17: Shelton, D. C, G. C. Anderson, T. S. Clark and C. E. Weakley, Jr., Studies on the vitamin K requirement of the chick. Poultry Sci. 35: Steel, R. G. D., and J. H. Torrie, Principles and Procedures of Statistics. McGraw-Hill Book Company, Inc., New York. Yacowitz, H., E. Ross, V. L. Sanger, E. N. Moore and R. D. Carter, Hemorrhagic syndrome in chicks fed normal rations supplemented with sulfaquinoxaline. Proc. Soc. Exptl. Biol. Med. : 1-6. The Incidence of Physically Abnormal Turkey Hatching Eggs and Their Relationship to Hatchability F. L. CHERMS Department of Poultry Science, University of Wisconsin, Madison, Wisconsin AND E. WOLFF Bellefontaine Farms, Belleville, Wisconsin (Received for publication September 8, 1967) INTRODUCTION hatchability, they did point out that MOST commercial turkey hatching t chalk-white and porous eggs have very low eggs have only about a 60 percent ; hatchability. They also reported that the chance of producing a healthy poult. The ; reasons for such a low reproductive response are many and varied. It is well 1 known, for example, that fertility as measured by most commercial hatcheries does 5 not approach 100 percent. The influence of [ a group of factors, classified here as physical abnormalities upon hatchability of turkey hatching eggs, has not been studied in i great detail. large calcareous deposits present on many eggs were not related to hatchability. Air cell abnormalities, as related to hatchability, have been studied only for chicken eggs. Knox and Olsen (1936) found that artificially produced tremulous air cells significantly reduced hatchability. Byerly and Haynes (1938) and Olsen and Haynes (1949) reported that tremulous air cells, free air cells, and misplaced air cells In a study of several physical measurements adversely affected hatchability. These of turkey eggs, Brunson and God frey (1953) reported that only extreme ; variations in shape were detrimental to 3 hatching. Shell thickness, as measured by j specific gravity, has been shown by Payne i and McDaniel (1958) to have an influence e on hatchability, since they reported that t more help-outs came from eggs of low to D workers also found that low hatchability resulted from misshapen eggs, cracks, and eggs with porous shells. The incidence of physically abnormal eggs has received slight attention. Byerly and Haynes (1938) found that 14.5 per- cent of the eggs in their study were rejected as hatching eggs. The breakdown of medium specific gravity. Although Phillips s some of their reject categories was as follows: and Williams (1944) reported a lack of relationship between specific gravity and i cracks 70 percent, porous shell 1.8 percent, misplaced air cell 1.3 per-