Protective Effect of Nicarbazin on Nutritional Encephalopathy in Chicks1 I. BARTOV and P. BUDOWSKI Division of Poultry Science, Agricultural Research Organization, The Volcani Center, Rehovot, Israel and Department of Animal Sciences, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot, Israel (Received for publication June 6, 1978)
1979 Poultry Science 58:597-601 INTRODUCTION Nutritional encephalopathy, usually referred to as encephalomalacia, may be induced experimentally in chicks by diets low in a-tocopherol and containing large amounts of linoleic acid (Dam et al, 1958; Century and Horwitt, 1959; Machlin and Gordon, 1960; Mokadi and Budowski, 1963; Bartov and Bornstein, 1972; Dror et al., 1976). These factors, however, are not necessarily involved in the causation of that disease under practical field conditions, since outbreaks of encephalopathy are still observed in spite of inclusion in the diets of sufficient vitamin E and synthetic antioxidants (Hislop and Whittle, 1967a,b; Marthedal, 1973), nor has it been possible to relate these outbreaks to deterioration of lipids in the feeds (Mokadi and Budowski, 1963). Dietary factors unrelated to an imbalance of dietary tocopherol and polyunsaturated fatty acids or to deterioration of lipids have occasionally been reported to induce encephalopathy under experimental conditions. This is the case for phytol (Pudelkiewicz et al., 1970) and for dilauryl succinate (Yoshida et al., 1971). We recently observed an outbreak of encephalopathy in broilers raised on litter and fed a diet containing the coccidiostat amprolium [l-(4-amino-
1
Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 159-E, 1978 series. 2 Folkman and Dr. Koffler Ltd., Tel Aviv, Israel. 3 Shemen Ltd., Haifa, Israel.
2 -n -propyl -5-pyrimidinylmethyl) -2 -picolinium chloride hydrochloride] 2 , while broilers from the same source but raised in cages and receiving the same diet without the coccidiostat were not affected. The purpose of the present experiments was to investigate whether dietary coccidiostats have any effect on the development of encephalopathy. EXPERIMENTAL METHODS Animals. Day-old crossbred New Hampshire X White Leghorn male chicks were used throughout the study. They were housed in electrically heated battery brooders equipped with raised wire floors. The birds had free access to water and were fed ad lib. Diets. The compositions of the basal diets are given in Table 1. Methyl esters were prepared from refined safflower oils as follows: safflower oil 3 was transmethylated by refluxing with an equal volume of methanol and .5% (w/v) sodium hydroxide pellets. The alkali was added in 3—4 portions over a period of 30 min. By that time the mixture became homogeneous. Heating under reflux was continued for another hour, after which the mixture was cooled. The alkali was neutralized with an excess of acetic acid and enough water was added to achieve separation of the methyl esters. The latter was washed with water, dried, and distilled over vacuum. Oxidized safflower oil with low tocopherol content was prepared by aeration at 145 C for 24 hr as described previously (Dror et al., 1976).
597
Downloaded from http://ps.oxfordjournals.org/ at Juridische Bibliotheek Der UU/University Library Utrecht on April 23, 2015
ABSTRACT Nutritional encephalopathy was induced in young chicks by vitamin E-deficient diets containing either 4% methyl esters of safflower oil or 10% thermally oxidized safflower oil. The coccidiostat nicarbazin (an equimolecular complex of 4,4'-dinitrocarbanilide and 2-hydroxy 4,6-dimethylpyrimidine) reduced the incidence of encephalopathy, but zoalene (3,5-dinitro-otoluamide) and amprolium [l-(4-amino-2-n-propyl-5-pyrimidinylmethyl)-2-picoliniurn chloride hydrochloride] did not. Neither of the two components of nicarbazin affected the rate of development of encephalopathy when fed separately, but when included together in the diet, they reduced the incidence of the disease.
598
BARTOV AND BUDOWSKI
TABLE 1. Percentage composition of the basal diets Diet A
52.00 38.36 4.00
56.00 26.35
1.00 4.00 .50 .14
10.00 3.00 4.00 .50 .15
Calculated analysis 23.54 Crude protein (%) Metabolizable energy (kcal/kg) 2758
25.35 2971
Supplying per kg feed: Dicalcium phosphate, 28 g; limestone, 7 g; salt, 3.5 g; manganese, 120 mg; zinc, 75 mg; iron, 37.5 mg; copper, 3 mg; iodine, 1.8 mg; cobalt, .3 mg. Supplying per kg feed: vitamin A, 3000 IU; vitamin D 3 , 200 1CU; menadione sodium bisulfite, 1.0 mg; thiamine, 3.6 mg; riboflavin, 7.2 mg; Ca-pantothenate, 20.0 mg; niacin, 55.0 mg; pyridoxine, 6.0 mg; biotin, .2 mg; choline, 1300 mg; folic acid, 2.4 mg; vitamin B, 2 , .02 mg; BHT, 50 mg.
Diet A, containing 4% methyl esters (Table 1), was used as the basal diet in trials 1 to 4. Each of the experimental diets in these trials was fed to a group of 25 one-day-old chicks, the number of which was reduced to 20 at the age of 7 days by removing the lightest chicks. The trials were terminated at the age of 21 days. However, body weights were determined only up to 14 days of age in order to exclude the effect of encephalopathy on this parameter. In trial 5, a combination of diets A and B (Table 1) was used as described below. The coccidiostats were usually incorporated into the basal diets at 250 mg/kg, i.e., twice the concentration recommended for the prevention of coccidiosis (details in Tables 2 to 5 and Fig. 1). In trials 1, 2, and 5, the commercial coccidiostats also contained ethopabate, hence the supplements also added 16 mg of this compound per kg diet. In trials 3 and 4, pure nicarbazin (an equimolecular complex of 4,4'dinitrocarbanilide [DNC] and 2-hydroxy 4,6dimethylpyrimidine [HDP]) and its constituent compounds 2 were used.
"Abie, Ltd., Ramat Gan, Israel.
RESULTS Trial 1. The purpose of this experiment was to compare the effect of amprolium,nicarbazin, and a third coccidiostat, zoalene (3,5-dinitro-otoluamide) 4 , on the severity of encephalopathy. The results are summarized in Table 2. Amprolium and zoalene did not affect the severity of the disease; however, nicarbazin significantly decreased it. Encephalopathy was completely prevented in a group of four chicks receiving the basal diet supplemented with 10 mg atocopheryl acetate/kg. Nicarbazin slightly decreased body weight at 14 days, before the onset of encephalopathy. Trial 2. This experiment was carried out in order to evaluate the effect of dietary nicarbazin concentration on its protective effect against encephalopathy. The results are given in
TABLE 2. Effect of various coccidiostats on the severity of encephalopathy after 21 days (trial I) Incidence of encephalopathy 6 Dietary additive 6 (mg/kg)
Body weight atl4daysd (g)
Ataxia
Mortality
None f Amprolium, 250 Zoak-ne, 250 Nicarbazin, 234
108 111 104 97
10 a 10 a 10* 2°
6 4 5 2
' Values within a column not having one letter in common, differ significantly (P.05). Trial 5. The purpose of this trial was to study the protective action of nicarbazin under a different dietary regime. Two groups of 25 chicks each were fed diet A (Table 1) up to the seventh day of age, then their number was reduced to 20 per group, and they were changed to the experimental diets based on diet B (10% oxidized safflower oil, Table 1) during the next 14 days. Results of the trial are presented in Figure 1. Nicarbazin had some protective effect which lasted ca. 7 days for ataxia and about 13 days with mortality as the criterion.
TABLE 4. Effect of dietary nicarbazin and its constituents on the incidence of encephalopathy at 21 days (trial 3) Incidence of encephalopathy'
Dietary additive (mg/kg)
Body weight at 14 days e (g)
Ataxia
Mortality
None Nicarbazin, 234 DNCS, 165.9 + HDPS, 68.1 DNC, 165.9 HDP, 68.1
126 115 126 121 121
lia.b 8 b .c 5C 16 a 15 a
6 b ,c id 2C,d oa,b 14a
' ' c ' Values within a column not having one letter in common, differ significantly (P
1979 Poultry Science 58:597-601 INTRODUCTION Nutritional encephalopathy, usually referred to as encephalomalacia, may be induced experimentally in chicks by diets low in a-tocopherol and containing large amounts of linoleic acid (Dam et al, 1958; Century and Horwitt, 1959; Machlin and Gordon, 1960; Mokadi and Budowski, 1963; Bartov and Bornstein, 1972; Dror et al., 1976). These factors, however, are not necessarily involved in the causation of that disease under practical field conditions, since outbreaks of encephalopathy are still observed in spite of inclusion in the diets of sufficient vitamin E and synthetic antioxidants (Hislop and Whittle, 1967a,b; Marthedal, 1973), nor has it been possible to relate these outbreaks to deterioration of lipids in the feeds (Mokadi and Budowski, 1963). Dietary factors unrelated to an imbalance of dietary tocopherol and polyunsaturated fatty acids or to deterioration of lipids have occasionally been reported to induce encephalopathy under experimental conditions. This is the case for phytol (Pudelkiewicz et al., 1970) and for dilauryl succinate (Yoshida et al., 1971). We recently observed an outbreak of encephalopathy in broilers raised on litter and fed a diet containing the coccidiostat amprolium [l-(4-amino-
1
Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 159-E, 1978 series. 2 Folkman and Dr. Koffler Ltd., Tel Aviv, Israel. 3 Shemen Ltd., Haifa, Israel.
2 -n -propyl -5-pyrimidinylmethyl) -2 -picolinium chloride hydrochloride] 2 , while broilers from the same source but raised in cages and receiving the same diet without the coccidiostat were not affected. The purpose of the present experiments was to investigate whether dietary coccidiostats have any effect on the development of encephalopathy. EXPERIMENTAL METHODS Animals. Day-old crossbred New Hampshire X White Leghorn male chicks were used throughout the study. They were housed in electrically heated battery brooders equipped with raised wire floors. The birds had free access to water and were fed ad lib. Diets. The compositions of the basal diets are given in Table 1. Methyl esters were prepared from refined safflower oils as follows: safflower oil 3 was transmethylated by refluxing with an equal volume of methanol and .5% (w/v) sodium hydroxide pellets. The alkali was added in 3—4 portions over a period of 30 min. By that time the mixture became homogeneous. Heating under reflux was continued for another hour, after which the mixture was cooled. The alkali was neutralized with an excess of acetic acid and enough water was added to achieve separation of the methyl esters. The latter was washed with water, dried, and distilled over vacuum. Oxidized safflower oil with low tocopherol content was prepared by aeration at 145 C for 24 hr as described previously (Dror et al., 1976).
597
Downloaded from http://ps.oxfordjournals.org/ at Juridische Bibliotheek Der UU/University Library Utrecht on April 23, 2015
ABSTRACT Nutritional encephalopathy was induced in young chicks by vitamin E-deficient diets containing either 4% methyl esters of safflower oil or 10% thermally oxidized safflower oil. The coccidiostat nicarbazin (an equimolecular complex of 4,4'-dinitrocarbanilide and 2-hydroxy 4,6-dimethylpyrimidine) reduced the incidence of encephalopathy, but zoalene (3,5-dinitro-otoluamide) and amprolium [l-(4-amino-2-n-propyl-5-pyrimidinylmethyl)-2-picoliniurn chloride hydrochloride] did not. Neither of the two components of nicarbazin affected the rate of development of encephalopathy when fed separately, but when included together in the diet, they reduced the incidence of the disease.
598
BARTOV AND BUDOWSKI
TABLE 1. Percentage composition of the basal diets Diet A
52.00 38.36 4.00
56.00 26.35
1.00 4.00 .50 .14
10.00 3.00 4.00 .50 .15
Calculated analysis 23.54 Crude protein (%) Metabolizable energy (kcal/kg) 2758
25.35 2971
Supplying per kg feed: Dicalcium phosphate, 28 g; limestone, 7 g; salt, 3.5 g; manganese, 120 mg; zinc, 75 mg; iron, 37.5 mg; copper, 3 mg; iodine, 1.8 mg; cobalt, .3 mg. Supplying per kg feed: vitamin A, 3000 IU; vitamin D 3 , 200 1CU; menadione sodium bisulfite, 1.0 mg; thiamine, 3.6 mg; riboflavin, 7.2 mg; Ca-pantothenate, 20.0 mg; niacin, 55.0 mg; pyridoxine, 6.0 mg; biotin, .2 mg; choline, 1300 mg; folic acid, 2.4 mg; vitamin B, 2 , .02 mg; BHT, 50 mg.
Diet A, containing 4% methyl esters (Table 1), was used as the basal diet in trials 1 to 4. Each of the experimental diets in these trials was fed to a group of 25 one-day-old chicks, the number of which was reduced to 20 at the age of 7 days by removing the lightest chicks. The trials were terminated at the age of 21 days. However, body weights were determined only up to 14 days of age in order to exclude the effect of encephalopathy on this parameter. In trial 5, a combination of diets A and B (Table 1) was used as described below. The coccidiostats were usually incorporated into the basal diets at 250 mg/kg, i.e., twice the concentration recommended for the prevention of coccidiosis (details in Tables 2 to 5 and Fig. 1). In trials 1, 2, and 5, the commercial coccidiostats also contained ethopabate, hence the supplements also added 16 mg of this compound per kg diet. In trials 3 and 4, pure nicarbazin (an equimolecular complex of 4,4'dinitrocarbanilide [DNC] and 2-hydroxy 4,6dimethylpyrimidine [HDP]) and its constituent compounds 2 were used.
"Abie, Ltd., Ramat Gan, Israel.
RESULTS Trial 1. The purpose of this experiment was to compare the effect of amprolium,nicarbazin, and a third coccidiostat, zoalene (3,5-dinitro-otoluamide) 4 , on the severity of encephalopathy. The results are summarized in Table 2. Amprolium and zoalene did not affect the severity of the disease; however, nicarbazin significantly decreased it. Encephalopathy was completely prevented in a group of four chicks receiving the basal diet supplemented with 10 mg atocopheryl acetate/kg. Nicarbazin slightly decreased body weight at 14 days, before the onset of encephalopathy. Trial 2. This experiment was carried out in order to evaluate the effect of dietary nicarbazin concentration on its protective effect against encephalopathy. The results are given in
TABLE 2. Effect of various coccidiostats on the severity of encephalopathy after 21 days (trial I) Incidence of encephalopathy 6 Dietary additive 6 (mg/kg)
Body weight atl4daysd (g)
Ataxia
Mortality
None f Amprolium, 250 Zoak-ne, 250 Nicarbazin, 234
108 111 104 97
10 a 10 a 10* 2°
6 4 5 2
' Values within a column not having one letter in common, differ significantly (P.05). Trial 5. The purpose of this trial was to study the protective action of nicarbazin under a different dietary regime. Two groups of 25 chicks each were fed diet A (Table 1) up to the seventh day of age, then their number was reduced to 20 per group, and they were changed to the experimental diets based on diet B (10% oxidized safflower oil, Table 1) during the next 14 days. Results of the trial are presented in Figure 1. Nicarbazin had some protective effect which lasted ca. 7 days for ataxia and about 13 days with mortality as the criterion.
TABLE 4. Effect of dietary nicarbazin and its constituents on the incidence of encephalopathy at 21 days (trial 3) Incidence of encephalopathy'
Dietary additive (mg/kg)
Body weight at 14 days e (g)
Ataxia
Mortality
None Nicarbazin, 234 DNCS, 165.9 + HDPS, 68.1 DNC, 165.9 HDP, 68.1
126 115 126 121 121
lia.b 8 b .c 5C 16 a 15 a
6 b ,c id 2C,d oa,b 14a
' ' c ' Values within a column not having one letter in common, differ significantly (P
