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Effect of Potassium Nitrate Intake on Dairy Calves1
E F F E C T OF P O T A S S I U M N I T R A T E I N T A K E ON D A I R Y C A L V E S 1 R. D. PREWITT AND C. P. MERILAN
Depart*nent of Dairy TJusbandry, University of Missouri, Coht,mbia SUMMARY
This study provides infornmtion on the effects of level, frequency, and duration of nitrate intake on the nitrate tolerance of young dairy animals. Administration of KNO~ to bull calves indicated that the minimum lethal dose for initial intake was in excess of 30 g. per 100 lb. body weight. Toxic response to the compound was found to vary between animals, but was not materially affected by experimental method of intake (by capsule or dissolved in milk). The level of KNO~ ingestion exerted little, if any, influence on the time required for the in vivo formation of maximal levels of methemoglobin (determined spectrophotometrically). Up to 1.3 g. % nitrate, but no nitrite, was detected by microbiological assay of the urine from two calves receiving 25 g. K N Q per 100 lb. body weight and showing low levels of methemoglobin.
D u r i n g recent years, nitrate poisoning has become increasingly important to the livestock industry, either by virtue of an increased incidence or owing to more accurate diagnosis of the symptoms. Most of the early work indicates that this condition was associated primarily with the ingestion of oat h a y or corn stalks high in KNOa equivalent ( 2 , 5 , 9 , 1 5 - 1 7 ) . More recently, other feed materials such as corn silage, sorgo silage, and alfalfa hay also have been implicated in nitrate poisoning of cattle and sheep, when the KNOa equivalent on a d r y matter basis exceeded approximately 0.7% of the feedstuff. Davidson et al. (5) reported that the symptoms of nitrate poisoning were quite uniform in character, and that the severity of these symptoms was markedly increased if the animals were disturbed, or forced to move about. The symptoms included an increased pulse rate, weakness, staggering gait, apparent blindness in some cases, and cyanosis; in severe cases, death usually occurred with little or no struggling. Similar symptoms were reported by Muhrer et al. (13), with the exception that motor spasms progressed rapidly to convulsions, terminating in death. However, in the latter report, nitrate was only one of a group of m a n y factors believed to be responsible for the extreme toxicity of forages produced in a drought area. Beath et al. (1) have reported similar symptoms. Ingested nitrates are believed to be converted to nitrites in the digestive tract (1, 12), and the nitrites then absorbed by the blood stream cause the hemoglobin to be converted into methemoglobin, which is incapable of carrying oxygen to the tissues (1, 6, 12, 13, 14). The methemoglobin will gradually be reduced to normal hemoglobin in drawn blood samples (6, 10). Relatively little information is available on the importance of nitrate poisoning in y o u n g animals and on its relationship to their later growth and development. However, B r a d l y et al. (4) report that the minimum lethal dose of KN03 Received for publication August 28, 1957. 1Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 1787. Approved by the Director. 807
808
R.D.
P R E W I T T AND C. P. I ~ E R I L A N
(by stomach tube) for dairy calves is approximately 25 g. per 100 lb. body weight, with large quantities of nitrite being found in the bile and urine of animals receiving the nitrates. METHODS
Bull calves from the University of Missouri dairy herd were used as experimental animals. The calves nursed their dams for the first three days after birth and were bucket-fed thereafter, receiving whole milk (10% of body weight daily) to the age of 1 too., followed by a gradual change to skimmilk at a daily rate of 10% of body weight, until a maximum of 14 lb. skimmilk was reached. Grain was fed free choice, with the older calves consuming approximately 4 lb. per day. Alfalfa hay, containing less than 0.21% KN03 equivalent on a dry matter basis, was fed ad libitum. Potassium nitrate dosages were administered either by gelatin capsule or by dissolving the compound in skimmilk. Blood samples were drawn from the jugular vein into sterile tubes containing 1.5 rag. potassium oxalate per milliliter of blood. Analysis for hemoglobin and methemoglobin was started within 30 rain. after the sample was drawn, since methemoglobin in stored blood is converted back slowly to functional hemoglobin (10). A Coleman Model 14 Spectrophotometer was used with the method of Evelyn and Malloy (7), for determining hemoglobin and methemoglobin. Urine samples were collected from a limited number of calves, using a modification of the urine-collection apparatus as designed by Hobbs et al. (11). Urine samples were held in a frozen state ( - 1 5 ° C.) between collection and analysis for nitrate and nitrite content. The microbiological method developed by Garner et al. (8) was used in determining the nitrate and nitrite content of the alfalfa hay and urine samples. OBSERVATIONS AND R E S U L T S
Individual animals vary markedly in the time that maximum conversion of hemoglobin to methemoglobin occurs following nitrate ingestion (Table 1). However, neither the level of nitrate intake nor the mode of intake (gelatin capsules vs. milk) appears to have much influence on the time of maximum conversion. The slight increase in methemoglobin formation with KN03 given in capsules may be, in part, owing to a carryover in toxic effect from the dosage given in milk during the preceding week. Most of the calves refused to drink milk contaiuing more than 1.9 g. % KNO~. In the experimental trials, maximum conversion occurred approximately 5 hr. after nitrate intake. In contrast to the 25-g. minimum lethal dose reported by Bradly et al. (4), no calves died in this series of feeding trials, even though doses up to 30 g. per 100 lb. body weight were given. A possible explanation for the difference in tolerance could be owing in part to the level of energy in the ration and to the condition of the calves at the time of feeding potassium nitrate.
809
E F F E C T OF N I T R A T E ON CALVES
The response (conversion of hemoglobin to methemoglobin) of calves to initial KNOs intake shows considerable individual variation, but also exhibits a general trend of increase in toxic symptoms with increasing KN03 dosage levels (Table 1). The maximum methemoglobin values at 10, 15, 20, and 30 g. per 100 lb. body weight dosages were 0, 8, 16, and 37%, respectively. In all cases where animals received nitrates, and showed methemoglobin formation, there was an increase in heart rate (Figure 1). 5 9 9 J x---x 3OG. KNO 3 / I O O L B . BODY WT. 380H ~ 2 0 G . KNO 3 / I O O L B , BODY w'r;
160 Lid
n't._ << bJbJ 120
10(3
80"
7C Z
6o
0 .j ,50 0 0~ ° 40
i,i "r f-w
30 20 IG 2 HOURS
FIG. 1. R e l a t i o n s h i p
4 AFTER
6
8
I0
KNO31NTAKE
between heart rate and methemoglobin
formation.
Large quantities of nitrates were found to be excreted in the urine of calves receiving 25 g. KN03 per 100 lb. body weight (Figure 2). However, no nitrites were detected in the urine at this level of KN03 intake. As shown in Figure 2, excretion of nitrates in the urine, and conversion of hemoglobin to methemoglobin in blood, occur at approximately the same time following K N Q ingestion. Two calves were given multiple doses of potassium nitrate (15 g. per 100 lb. body weight) over a period of eight days. The calf receiving a single daily KNOs dose gained an average of 2 lb. in body weight per day and showed less than 10% maximum conversion of hemoglobin to methemoglobin on any given day. The other calf, receiving a total of 30 g. per 100 lb. body weight daily, in two equal portions given 12 hr. apart, neither gained nor lost weight during the
SI0
R.D.
PREWITT
AND
C. P. 51ERILAN
TABLE
1
Time of ¢naximuqn hemoglobin-to-methemoglobin conversion folIowi~g potassi~m nitrate inta#e
Calf No.
492ti 380H 490H 460H 498H
Age
Body wt.
Dosage level
(w#.)
(lb.)
(g. K_YOs/cwt)
6 9 5 8 8 7 8 9 10
155 200 147 190 188 168 146 ]62 164 181 158 174 185 200 181 206 223 172 198 224 238
10 20 10 20 15 15 20 20 25 25 20 20 25 25 20 25 25 30 30 25 25
ii
4'95H
485H 599/[
9 l0 11 12 10 12 13 15 18 20 21
Method of KNO3 intake
Maximum conversion H B to M H B
H r . a f t e r intake when maximum conversion occurred
(%) Capsule Capsule Capsule Capsule Capsule Capsule Milk Capsule Milk Capsule Capsule Milk Milk Capsule Capsule Milk Capsule Capsule Capsule Milk Capsule
0 19 0 34 8 4 0 0 5 19 5 1 3 4 16 4 11 37 76 3 3
.... 6 .... 6 5 5 .... 4 8 5 7 4 4, 6 3 4 4 9 5 4, 6, 8 4
8
o6 hJ I
2
W
I
g~
14i
I
i
i
6
8
i
V._z 12 ~rf
"'rE 6
2~
2 2
HOURS
4
AFTER
KNO 3 INTAKE
FIG. 2. U r i n a r y n i t r a t e excretion and methemoglobin f o r m a t i o n in two calves receiving 25 g. K N O J l 0 0 lb. body weight,
EFFECT
OF
NITRATE
ON
CALVES
811
e i g h t - d a y e x p e r i m e n t . The l a t t e r calf showed m a x i m u m hemoglobin-to-meth e m o g l o b i n c o n v e r s i o n values of b e t w e e n 35 a n d 50% on f o u r of the eight days. The toxic s y m p t o m s observed t h r o u g h o u t this s t u d y , for both the single a n d m u l t i p l e K N 0 3 doses, were r e l a t i v e l y mild, as c o m p a r e d to n i t r a t e p o i s o n i n g s y m p t o m s g e n e r a l l y seen both i n d a i r y a n d i n beef cattle u n d e r p r a c t i c a l f a r m conditions, at m u c h lower levels of n i t r a t e intake. This i m m e d i a t e l y poses a q u e s t i o n as to the t r u e n a t u r e of so-called n i t r a t e p o i s o n i n g observed on the f a r m . The a p p a r e n t d i s c r e p a n c y b e t w e e n s y m p t o m s i n d u c e d e x p e r i m e n t a l l y a n d those observed u n d e r p r a c t i c a l c o n d i t i o n s m a y be o w i n g either e n t i r e l y or i n p a r t to: ( 1 ) F o r m of n i t r a t e i n g e s t e d ( i n o r g a n i c vs. o r g a n i c ) ; ( 2 ) diet, r u m e n f u n c t i o n , a n d n u t r i t i o n a l s t a t u s of a n i m a l s i n c o n j u n c t i o n w i t h a m o u n t of e n v i r o n m e n t a l stress; ( 3 ) age a n d factors associated w i t h stress of milk a n d m e a t p r o d u c t i o n , a n d ( 4 ) level of n i t r a t e intake, p a r t i c u l a r l y as r e l a t e d to t o t a l p e r i o d of i n t a k e a n d a n m u n t c o n s u m e d at a n y g i v e n time. REFERENCES (1) BreATH,O. A., BILBEa, C. S., EPPSON, H. F., AND ROSEiXrFE'LD,h~ENE. Poisonous Plants and Livestock Poisoning. Wyoming Agr. Expt. Sta., Bull. 324. 1953. (2) BRADLY, W. B., ]~E,ATH, O. A., A~N'DEPPSON, H. F. Oat Hay Poisoning. Science, 89: 365. 1939. (3) BRADI,Y, W. B., EPPSO~T, H. F., AND BEATI-I, O. A. Nitrate as the Cause of Oat Hay Poisoning. J. A ~ . Vet. Med. Assoc., 94: 54]. 1939. (4) I~I~ADLY,W. B., EPPSON, H. F., AND Blg_a~I-I,O. A. Livestock Poisoning by Oat Hay and Other Plants Containing Nitrates. Wyoming Agr. Expt. Sta., Bull. 241. 1940. (5) DAVIDSOH,W. B., DOUGHTY, J. L., AND BOLTOIg, J. L. Nitrate Poisoning of Livestock. Can. J. Comp. Med., 5: 303. 1941. (6) DUKES, H. t{, Physiology of Domestic A~i~als. 7th ed. Comstock Publ. Assoc., Ithaca, N . Y . 1955. (7) EVI~LYN, K. A., AND MALLOY, H. T. Spectrophotometric Determination of Methemoglobin and Total Hemoglobin. J. Biol. Chem., 125: 655. 1938. (8) GARNXa~,G. B., BAU)aSTARK,J. S., MU~Ir~ER,M. E., AND PFANI)ER,W. H. Microbiological Determination of Nitrate. Anal. Chem., 28: 1589. 1956. (9) GILBI~RT~C. S., EPPSOH, H. F., BI~ADLY, W. B., AND BEATH, O. A. Nitrate Accumulation in Cultivated Plants and Weeds. Wyoming Agr. Expt. Sta., Bull. 277. 1946. (10) HEUBNF±I~,W., AND STUHLI~L~NN,M. Formation of Methemoglobin. XXI. Reversion in Drawn Blood. Arch. exptl. Pathol. Pharmakol., 199: 1. 1942. (11) HOBBS, C. S., HANSARD, S. L., ANn BAltltICK, E. 1~. Simplified Methods and Equipment Used in Separation of Urine from Feces Eliminated by Heifers and Steers. J. Animal Sci., 9: 565. 1950. (12) MONR@E, C. F., AND PE~RKINS, A. E. n Study of the pH Values of the Ingesta of the Bovine Rumen. J. Dairy Sci., 22: 983. 1939. (13) MUHI~EIZ~M. E., CASE;,A. A., GAIINE~R,G. B., AND PFANDER, W. H. Toxic Forage Produced in a Drought Area. J. Animal Sci., 14: 1251. 1955. (14) MUH~I~, M. E., .~ND P~'ANI)E~, W. H. Forage Poisoning in Missouri Due to Excessive Amounts of Nitrate. Missouri Agr. Expt. Sta., !lesearch Bull. 652. pp. 19. 1955. (15) NE'WSON, I. E., SCOUT) E. N., THORP, F., JR., BAlkER, C. W., AND GROTH, A. H. Oat Hay Poisoning. J. Am. Vet. Med. Assoc., 90: 66. 1937. (16) OLSON, O. E., A~'qD WHITEHE'AD, E. Nitrate Content of Some South Dakota Plants. Proc. S. Dakota Acad. Sci., 20: 95. 1940. (17) TH01ZP, F., Jl% Further Observations on Oat Hay Poisoning. J. Am. Vet. Med. Assoc., 4 5 : 159. 1938.
Depart*nent of Dairy TJusbandry, University of Missouri, Coht,mbia SUMMARY
This study provides infornmtion on the effects of level, frequency, and duration of nitrate intake on the nitrate tolerance of young dairy animals. Administration of KNO~ to bull calves indicated that the minimum lethal dose for initial intake was in excess of 30 g. per 100 lb. body weight. Toxic response to the compound was found to vary between animals, but was not materially affected by experimental method of intake (by capsule or dissolved in milk). The level of KNO~ ingestion exerted little, if any, influence on the time required for the in vivo formation of maximal levels of methemoglobin (determined spectrophotometrically). Up to 1.3 g. % nitrate, but no nitrite, was detected by microbiological assay of the urine from two calves receiving 25 g. K N Q per 100 lb. body weight and showing low levels of methemoglobin.
D u r i n g recent years, nitrate poisoning has become increasingly important to the livestock industry, either by virtue of an increased incidence or owing to more accurate diagnosis of the symptoms. Most of the early work indicates that this condition was associated primarily with the ingestion of oat h a y or corn stalks high in KNOa equivalent ( 2 , 5 , 9 , 1 5 - 1 7 ) . More recently, other feed materials such as corn silage, sorgo silage, and alfalfa hay also have been implicated in nitrate poisoning of cattle and sheep, when the KNOa equivalent on a d r y matter basis exceeded approximately 0.7% of the feedstuff. Davidson et al. (5) reported that the symptoms of nitrate poisoning were quite uniform in character, and that the severity of these symptoms was markedly increased if the animals were disturbed, or forced to move about. The symptoms included an increased pulse rate, weakness, staggering gait, apparent blindness in some cases, and cyanosis; in severe cases, death usually occurred with little or no struggling. Similar symptoms were reported by Muhrer et al. (13), with the exception that motor spasms progressed rapidly to convulsions, terminating in death. However, in the latter report, nitrate was only one of a group of m a n y factors believed to be responsible for the extreme toxicity of forages produced in a drought area. Beath et al. (1) have reported similar symptoms. Ingested nitrates are believed to be converted to nitrites in the digestive tract (1, 12), and the nitrites then absorbed by the blood stream cause the hemoglobin to be converted into methemoglobin, which is incapable of carrying oxygen to the tissues (1, 6, 12, 13, 14). The methemoglobin will gradually be reduced to normal hemoglobin in drawn blood samples (6, 10). Relatively little information is available on the importance of nitrate poisoning in y o u n g animals and on its relationship to their later growth and development. However, B r a d l y et al. (4) report that the minimum lethal dose of KN03 Received for publication August 28, 1957. 1Contribution from the Missouri Agricultural Experiment Station, Journal Series No. 1787. Approved by the Director. 807
808
R.D.
P R E W I T T AND C. P. I ~ E R I L A N
(by stomach tube) for dairy calves is approximately 25 g. per 100 lb. body weight, with large quantities of nitrite being found in the bile and urine of animals receiving the nitrates. METHODS
Bull calves from the University of Missouri dairy herd were used as experimental animals. The calves nursed their dams for the first three days after birth and were bucket-fed thereafter, receiving whole milk (10% of body weight daily) to the age of 1 too., followed by a gradual change to skimmilk at a daily rate of 10% of body weight, until a maximum of 14 lb. skimmilk was reached. Grain was fed free choice, with the older calves consuming approximately 4 lb. per day. Alfalfa hay, containing less than 0.21% KN03 equivalent on a dry matter basis, was fed ad libitum. Potassium nitrate dosages were administered either by gelatin capsule or by dissolving the compound in skimmilk. Blood samples were drawn from the jugular vein into sterile tubes containing 1.5 rag. potassium oxalate per milliliter of blood. Analysis for hemoglobin and methemoglobin was started within 30 rain. after the sample was drawn, since methemoglobin in stored blood is converted back slowly to functional hemoglobin (10). A Coleman Model 14 Spectrophotometer was used with the method of Evelyn and Malloy (7), for determining hemoglobin and methemoglobin. Urine samples were collected from a limited number of calves, using a modification of the urine-collection apparatus as designed by Hobbs et al. (11). Urine samples were held in a frozen state ( - 1 5 ° C.) between collection and analysis for nitrate and nitrite content. The microbiological method developed by Garner et al. (8) was used in determining the nitrate and nitrite content of the alfalfa hay and urine samples. OBSERVATIONS AND R E S U L T S
Individual animals vary markedly in the time that maximum conversion of hemoglobin to methemoglobin occurs following nitrate ingestion (Table 1). However, neither the level of nitrate intake nor the mode of intake (gelatin capsules vs. milk) appears to have much influence on the time of maximum conversion. The slight increase in methemoglobin formation with KN03 given in capsules may be, in part, owing to a carryover in toxic effect from the dosage given in milk during the preceding week. Most of the calves refused to drink milk contaiuing more than 1.9 g. % KNO~. In the experimental trials, maximum conversion occurred approximately 5 hr. after nitrate intake. In contrast to the 25-g. minimum lethal dose reported by Bradly et al. (4), no calves died in this series of feeding trials, even though doses up to 30 g. per 100 lb. body weight were given. A possible explanation for the difference in tolerance could be owing in part to the level of energy in the ration and to the condition of the calves at the time of feeding potassium nitrate.
809
E F F E C T OF N I T R A T E ON CALVES
The response (conversion of hemoglobin to methemoglobin) of calves to initial KNOs intake shows considerable individual variation, but also exhibits a general trend of increase in toxic symptoms with increasing KN03 dosage levels (Table 1). The maximum methemoglobin values at 10, 15, 20, and 30 g. per 100 lb. body weight dosages were 0, 8, 16, and 37%, respectively. In all cases where animals received nitrates, and showed methemoglobin formation, there was an increase in heart rate (Figure 1). 5 9 9 J x---x 3OG. KNO 3 / I O O L B . BODY WT. 380H ~ 2 0 G . KNO 3 / I O O L B , BODY w'r;
160 Lid
n't._ << bJbJ 120
10(3
80"
7C Z
6o
0 .j ,50 0 0~ ° 40
i,i "r f-w
30 20 IG 2 HOURS
FIG. 1. R e l a t i o n s h i p
4 AFTER
6
8
I0
KNO31NTAKE
between heart rate and methemoglobin
formation.
Large quantities of nitrates were found to be excreted in the urine of calves receiving 25 g. KN03 per 100 lb. body weight (Figure 2). However, no nitrites were detected in the urine at this level of KN03 intake. As shown in Figure 2, excretion of nitrates in the urine, and conversion of hemoglobin to methemoglobin in blood, occur at approximately the same time following K N Q ingestion. Two calves were given multiple doses of potassium nitrate (15 g. per 100 lb. body weight) over a period of eight days. The calf receiving a single daily KNOs dose gained an average of 2 lb. in body weight per day and showed less than 10% maximum conversion of hemoglobin to methemoglobin on any given day. The other calf, receiving a total of 30 g. per 100 lb. body weight daily, in two equal portions given 12 hr. apart, neither gained nor lost weight during the
SI0
R.D.
PREWITT
AND
C. P. 51ERILAN
TABLE
1
Time of ¢naximuqn hemoglobin-to-methemoglobin conversion folIowi~g potassi~m nitrate inta#e
Calf No.
492ti 380H 490H 460H 498H
Age
Body wt.
Dosage level
(w#.)
(lb.)
(g. K_YOs/cwt)
6 9 5 8 8 7 8 9 10
155 200 147 190 188 168 146 ]62 164 181 158 174 185 200 181 206 223 172 198 224 238
10 20 10 20 15 15 20 20 25 25 20 20 25 25 20 25 25 30 30 25 25
ii
4'95H
485H 599/[
9 l0 11 12 10 12 13 15 18 20 21
Method of KNO3 intake
Maximum conversion H B to M H B
H r . a f t e r intake when maximum conversion occurred
(%) Capsule Capsule Capsule Capsule Capsule Capsule Milk Capsule Milk Capsule Capsule Milk Milk Capsule Capsule Milk Capsule Capsule Capsule Milk Capsule
0 19 0 34 8 4 0 0 5 19 5 1 3 4 16 4 11 37 76 3 3
.... 6 .... 6 5 5 .... 4 8 5 7 4 4, 6 3 4 4 9 5 4, 6, 8 4
8
o6 hJ I
2
W
I
g~
14i
I
i
i
6
8
i
V._z 12 ~rf
"'rE 6
2~
2 2
HOURS
4
AFTER
KNO 3 INTAKE
FIG. 2. U r i n a r y n i t r a t e excretion and methemoglobin f o r m a t i o n in two calves receiving 25 g. K N O J l 0 0 lb. body weight,
EFFECT
OF
NITRATE
ON
CALVES
811
e i g h t - d a y e x p e r i m e n t . The l a t t e r calf showed m a x i m u m hemoglobin-to-meth e m o g l o b i n c o n v e r s i o n values of b e t w e e n 35 a n d 50% on f o u r of the eight days. The toxic s y m p t o m s observed t h r o u g h o u t this s t u d y , for both the single a n d m u l t i p l e K N 0 3 doses, were r e l a t i v e l y mild, as c o m p a r e d to n i t r a t e p o i s o n i n g s y m p t o m s g e n e r a l l y seen both i n d a i r y a n d i n beef cattle u n d e r p r a c t i c a l f a r m conditions, at m u c h lower levels of n i t r a t e intake. This i m m e d i a t e l y poses a q u e s t i o n as to the t r u e n a t u r e of so-called n i t r a t e p o i s o n i n g observed on the f a r m . The a p p a r e n t d i s c r e p a n c y b e t w e e n s y m p t o m s i n d u c e d e x p e r i m e n t a l l y a n d those observed u n d e r p r a c t i c a l c o n d i t i o n s m a y be o w i n g either e n t i r e l y or i n p a r t to: ( 1 ) F o r m of n i t r a t e i n g e s t e d ( i n o r g a n i c vs. o r g a n i c ) ; ( 2 ) diet, r u m e n f u n c t i o n , a n d n u t r i t i o n a l s t a t u s of a n i m a l s i n c o n j u n c t i o n w i t h a m o u n t of e n v i r o n m e n t a l stress; ( 3 ) age a n d factors associated w i t h stress of milk a n d m e a t p r o d u c t i o n , a n d ( 4 ) level of n i t r a t e intake, p a r t i c u l a r l y as r e l a t e d to t o t a l p e r i o d of i n t a k e a n d a n m u n t c o n s u m e d at a n y g i v e n time. REFERENCES (1) BreATH,O. A., BILBEa, C. S., EPPSON, H. F., AND ROSEiXrFE'LD,h~ENE. Poisonous Plants and Livestock Poisoning. Wyoming Agr. Expt. Sta., Bull. 324. 1953. (2) BRADLY, W. B., ]~E,ATH, O. A., A~N'DEPPSON, H. F. Oat Hay Poisoning. Science, 89: 365. 1939. (3) BRADI,Y, W. B., EPPSO~T, H. F., AND BEATI-I, O. A. Nitrate as the Cause of Oat Hay Poisoning. J. A ~ . Vet. Med. Assoc., 94: 54]. 1939. (4) I~I~ADLY,W. B., EPPSON, H. F., AND Blg_a~I-I,O. A. Livestock Poisoning by Oat Hay and Other Plants Containing Nitrates. Wyoming Agr. Expt. Sta., Bull. 241. 1940. (5) DAVIDSOH,W. B., DOUGHTY, J. L., AND BOLTOIg, J. L. Nitrate Poisoning of Livestock. Can. J. Comp. Med., 5: 303. 1941. (6) DUKES, H. t{, Physiology of Domestic A~i~als. 7th ed. Comstock Publ. Assoc., Ithaca, N . Y . 1955. (7) EVI~LYN, K. A., AND MALLOY, H. T. Spectrophotometric Determination of Methemoglobin and Total Hemoglobin. J. Biol. Chem., 125: 655. 1938. (8) GARNXa~,G. B., BAU)aSTARK,J. S., MU~Ir~ER,M. E., AND PFANI)ER,W. H. Microbiological Determination of Nitrate. Anal. Chem., 28: 1589. 1956. (9) GILBI~RT~C. S., EPPSOH, H. F., BI~ADLY, W. B., AND BEATH, O. A. Nitrate Accumulation in Cultivated Plants and Weeds. Wyoming Agr. Expt. Sta., Bull. 277. 1946. (10) HEUBNF±I~,W., AND STUHLI~L~NN,M. Formation of Methemoglobin. XXI. Reversion in Drawn Blood. Arch. exptl. Pathol. Pharmakol., 199: 1. 1942. (11) HOBBS, C. S., HANSARD, S. L., ANn BAltltICK, E. 1~. Simplified Methods and Equipment Used in Separation of Urine from Feces Eliminated by Heifers and Steers. J. Animal Sci., 9: 565. 1950. (12) MONR@E, C. F., AND PE~RKINS, A. E. n Study of the pH Values of the Ingesta of the Bovine Rumen. J. Dairy Sci., 22: 983. 1939. (13) MUHI~EIZ~M. E., CASE;,A. A., GAIINE~R,G. B., AND PFANDER, W. H. Toxic Forage Produced in a Drought Area. J. Animal Sci., 14: 1251. 1955. (14) MUH~I~, M. E., .~ND P~'ANI)E~, W. H. Forage Poisoning in Missouri Due to Excessive Amounts of Nitrate. Missouri Agr. Expt. Sta., !lesearch Bull. 652. pp. 19. 1955. (15) NE'WSON, I. E., SCOUT) E. N., THORP, F., JR., BAlkER, C. W., AND GROTH, A. H. Oat Hay Poisoning. J. Am. Vet. Med. Assoc., 90: 66. 1937. (16) OLSON, O. E., A~'qD WHITEHE'AD, E. Nitrate Content of Some South Dakota Plants. Proc. S. Dakota Acad. Sci., 20: 95. 1940. (17) TH01ZP, F., Jl% Further Observations on Oat Hay Poisoning. J. Am. Vet. Med. Assoc., 4 5 : 159. 1938.