Oxytetracycline in B o v i n e P l a s m a , M i l k , a n d U r i n e after Intrauterine Administration1 G. E. MILLER 2 and G. P. BERGT Department of Animal Science University of Minnesota St. Paul 55101, and Wendt Laboratories Belle Plaine, MN 56011 ABSTRACT
Six healthy lactating cows with normal but varying stages of estrous cycles were treated with chelated or nonchelated oxytetracycline by intrauterine infusion, 4 mg/kg body weight. The chelated drug was a mixture of special metal halides and oxytetracycline while the nonchelated form was a commercial oxytetracycline solution suitable for intramuscular or intravenous administration. Oxytetracycline concentrations in plasma, milk, and urine were determined at timed intervals following treatment. Both forms of the drug appeared in the plasma, but the chelated form was a small amount. The nonchelated oxytetracycline also appeared in the milk. However, no oxytetracycline was in milk of cows treated with chelated oxytetracycline. Both forms of the drug were excreted in the urine. Milk should be withheld from market 24 and 0 h after administration o f nonchelated and chelated forms. INTRODUCTION
Passage of drugs across the mammary gland, salivary gland, rumen and uterine membranes is being studied in the bovine. The penetration of drugs into body tissues depends on the transport mechanism across body membranes. Passage usually occurs by passive (nonionic) diffusion or by an active transport process (11). The movement of drugs from the bovine uterus into the blood has been studied to a limited degree (2, 3). Kendrick and Pier (9) administered a combination of penicillin and dihydrostreptomycin as well as oxytetracycline
Received February 4, 1974. Scientific Paper No. 8556, Minnesota Agricultural Experiment Station, St. Paul. 2Deceased.
into the uterus and determined the time these drugs remained in the milk after treatment of the cow. Movement of many sulfonamides in solution from the uterus into blood plasma, milk, and urine has been studied (4, 7, 8, 10, 13). Work of Miller et al. (14) indicated that the mechanism of movement of sulfamethazine (pK a 7.37) and sulfacetamide (pK a 5.37) across the uterine membrane of the cow was via nonionic diffusion. These studies have increased our knowledge o f the movement of drugs in body fluids, but many questions remain unanswered. The objective of this investigation was to measure differences between chelated and nonchelated oxytetracycline in rate and time of passage through tissue, their concentration in tissue, and the time required for their complete elimination from the milk of treated cows. This work has been in abstract (12). Oxytetracycline in solution is being used to treat uterine infections in cows. EXPERIMENTAL PROCEDURE
Six healthy lactating dairy cows with normal but varying stages of estrous cycles were treated with nonchelated or chelated oxytetracycline by intrauterine infusion. The treatment dose was 4 mg/kg body weight. The chelated drug was a mixture of special metal halides and oxytetracycline while the nonchelated form was a commercial oxytetracycline solution suitable for intramuscular or intravenous administration. Oxytetracycline activity was determined on the drug used to be sure proper doses were administered. A rubber conduit was placed over the vaginal orifice to collect urine and uterine fluid discharged through the cervix. Oxytetracycline was administered one time to each cow. Blood, milk, and urine samples were collected at .5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h after drug administration. Blood was centrifuged immediately, and the plasma and milk were frozen promptly. Urine volumes were 315
316
MILLER AND BERGT
measured, and an aliquot was frozen. A known amount of oxytetracycline was dissolved in plasma, milk, and urine, and frozen, thus enabling calculation of a degradation curve due to storage. Oxytetracycline activity was determined by bacteriological bioassay (6). RESULTS A N D DISCUSSION
Table 1 shows the average plasma concentration of oxytetracycline in six cows after intrauterine administration of a nonchelated form at 4 mg/kg body weight. Ox3rcetracycline was in plasma at the .5-h collection and remained through the 24-h collection in most cows. Peaks in plasma appeared in the 2-h sample. Samples from one cow showed no oxytetracycline in the plasma after the .5-h collection. Much of the drug in this cow must have been excreted via the cervix soon after treatment. Table 1 also depicts the average concentration of oxytetracycline in milk from six cows. The nonchelated form of oxytetracycline entered the milk in significant quantities. Oxytetracycline appeared at 4-h and remained through the 24-h collection. The plasma of two cows was low in antibiotic, and milk was below the minimal activity detected by this method (.05 /Jg/ml). Peak oxytetracycline activity in the milk appeared at the 12-h collection.
TABLE 1. Average oxytetracycline concentration in plasma and milk of six cows after intrauterine administration of 4 mg/kg body weight of chelated and nonchelated form (ug/ml). Hours after administration N .5
Chelated form Plasma Milk
Nonchelated form Plasma Milk
0
0
0
0 0
.21 .30 .39
.38 .35 .32 .18 .07
0
.03
0 0 0
4 6
.16 .11
0 0
8
.16
0
12 24 48 72
.12 0
0 0
0
0
0
0
0
0
0
0
96
0
0
0
0
120
0
0
0
0
1 2
Journal of Dairy Science Vol. 59, No. 2
0 0 0
Additionally, Table 1 shows average concentrations in plasma and milk of oxytetracycline in six cows after intrauterine administration of a chelated form at 4 mg/kg body weight. Four of the cows showed oxytetracycline activity in plasma. Peak concentration was lower in plasma of cows given chelated oxytetracycline than in those given nonchelated drug. Oxytetracycline activity appeared in 2-h and remained until the 12-h collection. Peak activity appeared at the 4-h collection. Two cows did not show any oxytetracycline in plasma. This may have been due to much of the drug being excreted via the cervix shortly after intrauterine treatment. No oxytetracycline activity appeared in the rrfilk of any of the cows after intrauterine administration of chelated oxytetracycline at 4 mg/kg body weight. Therefore, the chelated form may be the drug of choice for treating uterine infections in lactating cows. These average oxytetracycline concentrations (Table 1) suggest that milk from cows ~reated with the nonchelated form should be withheld at least 24 h while the milk from animals receiving the chelated form would not require withholding. The nonchelated form of oxytetracycline appeared in the milk at 4-h and remained through 24-h. This did not agree with Kendrick and Pier (9) who showed that there was no oxytetracycline in milk at 12, 24, 48, and 72-h posttreatment. They, however, used the rapid disc assay for penicillin activity (1) to determine the amount of oxytetracycline in the milk, an incorrect method. The chelated form of oxytetracycline crossed the uterine membrane into plasma and milk less completely than did the nonchelated form. This may have been due to the chelation of the oxytetracycline to the metal halides in the drug mixture. Therefore, this drug complex moved across the uterine membrane more slowly. REFERENCES
.04 .09 .14 .14 .10
1 Arret, B., and A. Kirschbaum. 1959. A rapid disc assay method for detecting penicillin in milk. J. Milk Food Technol. 329. 2 Bierschwal, C. J., and A. W. Uren. 1955. The absorption of sulfamethazine by the bovine uterus. J. Amer. Vet. Med. Ass. 126:398. 3 Bierschwal, C. J., and A. W. Uren. 1956. The absorption of chlortetracycline (Aureomycin) by the bovine uterus. J. Amer. Vet. Med. Ass. 129:373.
TECHNICAL NOTE 4 Bratton, A. C., and E. K. MarshaU. 1939. A new coupling c o m p o n e n t for sulfanilamide determination. J. Biol. Chem. 128:537. 5 Edman, L., O. Holmberg, I. Settergren, a n d C. B. Thorell. 1965. Resorption o f iodine in Lugol's solution and in an iodophos from the u t e r u s of cows. Nord. Vet. Med. 17:391. 6 Food a n d Drug Administration. 1968. Antibiotic residues in milk, dairy p r o d u c t s a n d animal tissues: Methods, reports and protocols. Guideline pamphlet on antibiotic analysis. 7 Huang, J., D. Hawkinson, and G. E. Miller. 1972. Passage o f Sulfadimethoxine from the bovine uterus into blood, milk, and urine. J. Dairy Sci. 55:705. (Abstr.) 8 Huang, J., a n d G. E. Miller. 1972. Mechanism of m o v e m e n t of sulfanilamide, sulfaethoxypyridazine, sulfamethazine, sulfadimethoxine, sulfacetamide, and salicylic acid across t h e uterine m e m b r a n e of the guinea pig. J. Dairy Sci. 55:705. (Abstr.) 9 Kendrick, J. W., and A. C. Pier. 1960. Antibiotic
10
11
12
13
14
317
levels in milk following intrauterine infusion. The California Vet., M a y - J u n e . Miller, G. E. 1971. Passage of drugs across the bovine uterine m e m b r a n e . Vet. Med. Rep., Agr. Ext. Serv. No. 4, 41, June. Miller, G. E., N. C. Banerjee, and C. M. Stowe, Jr. 1967. Drug m o v e m e n t between bovine milk and plasma as affected by milk pH. J. Dairy Sci. 50:1395. Miller, G. E., G. P. Bergt, W. M. Rose, D. B. Brunson, and R. G. Messer. 1973. Distribution of oxytetracycline in bovine plasma, milk, a n d urine following its intrauterine administration in two forms. J. Dairy Sci. 56:659. (Abstr.) Miller, G. E., and G. Rouse. 1970. Passage of drugs from the bovine uterus into blood, milk and urine. J. Dairy Sci. 53:652. (Abstr.) Miller, G. E., G. Rouse, a n d M. L. Fahning. 1971. Mechanism o f m o v e m e n t of sulfamethazine and sulfacetamide across the bovine uterine m e m b r a n e . J. Dairy Sci. 54:795. (Abstr.)
Journal of Dairy Science Vol. 59, No. 2
Six healthy lactating cows with normal but varying stages of estrous cycles were treated with chelated or nonchelated oxytetracycline by intrauterine infusion, 4 mg/kg body weight. The chelated drug was a mixture of special metal halides and oxytetracycline while the nonchelated form was a commercial oxytetracycline solution suitable for intramuscular or intravenous administration. Oxytetracycline concentrations in plasma, milk, and urine were determined at timed intervals following treatment. Both forms of the drug appeared in the plasma, but the chelated form was a small amount. The nonchelated oxytetracycline also appeared in the milk. However, no oxytetracycline was in milk of cows treated with chelated oxytetracycline. Both forms of the drug were excreted in the urine. Milk should be withheld from market 24 and 0 h after administration o f nonchelated and chelated forms. INTRODUCTION
Passage of drugs across the mammary gland, salivary gland, rumen and uterine membranes is being studied in the bovine. The penetration of drugs into body tissues depends on the transport mechanism across body membranes. Passage usually occurs by passive (nonionic) diffusion or by an active transport process (11). The movement of drugs from the bovine uterus into the blood has been studied to a limited degree (2, 3). Kendrick and Pier (9) administered a combination of penicillin and dihydrostreptomycin as well as oxytetracycline
Received February 4, 1974. Scientific Paper No. 8556, Minnesota Agricultural Experiment Station, St. Paul. 2Deceased.
into the uterus and determined the time these drugs remained in the milk after treatment of the cow. Movement of many sulfonamides in solution from the uterus into blood plasma, milk, and urine has been studied (4, 7, 8, 10, 13). Work of Miller et al. (14) indicated that the mechanism of movement of sulfamethazine (pK a 7.37) and sulfacetamide (pK a 5.37) across the uterine membrane of the cow was via nonionic diffusion. These studies have increased our knowledge o f the movement of drugs in body fluids, but many questions remain unanswered. The objective of this investigation was to measure differences between chelated and nonchelated oxytetracycline in rate and time of passage through tissue, their concentration in tissue, and the time required for their complete elimination from the milk of treated cows. This work has been in abstract (12). Oxytetracycline in solution is being used to treat uterine infections in cows. EXPERIMENTAL PROCEDURE
Six healthy lactating dairy cows with normal but varying stages of estrous cycles were treated with nonchelated or chelated oxytetracycline by intrauterine infusion. The treatment dose was 4 mg/kg body weight. The chelated drug was a mixture of special metal halides and oxytetracycline while the nonchelated form was a commercial oxytetracycline solution suitable for intramuscular or intravenous administration. Oxytetracycline activity was determined on the drug used to be sure proper doses were administered. A rubber conduit was placed over the vaginal orifice to collect urine and uterine fluid discharged through the cervix. Oxytetracycline was administered one time to each cow. Blood, milk, and urine samples were collected at .5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h after drug administration. Blood was centrifuged immediately, and the plasma and milk were frozen promptly. Urine volumes were 315
316
MILLER AND BERGT
measured, and an aliquot was frozen. A known amount of oxytetracycline was dissolved in plasma, milk, and urine, and frozen, thus enabling calculation of a degradation curve due to storage. Oxytetracycline activity was determined by bacteriological bioassay (6). RESULTS A N D DISCUSSION
Table 1 shows the average plasma concentration of oxytetracycline in six cows after intrauterine administration of a nonchelated form at 4 mg/kg body weight. Ox3rcetracycline was in plasma at the .5-h collection and remained through the 24-h collection in most cows. Peaks in plasma appeared in the 2-h sample. Samples from one cow showed no oxytetracycline in the plasma after the .5-h collection. Much of the drug in this cow must have been excreted via the cervix soon after treatment. Table 1 also depicts the average concentration of oxytetracycline in milk from six cows. The nonchelated form of oxytetracycline entered the milk in significant quantities. Oxytetracycline appeared at 4-h and remained through the 24-h collection. The plasma of two cows was low in antibiotic, and milk was below the minimal activity detected by this method (.05 /Jg/ml). Peak oxytetracycline activity in the milk appeared at the 12-h collection.
TABLE 1. Average oxytetracycline concentration in plasma and milk of six cows after intrauterine administration of 4 mg/kg body weight of chelated and nonchelated form (ug/ml). Hours after administration N .5
Chelated form Plasma Milk
Nonchelated form Plasma Milk
0
0
0
0 0
.21 .30 .39
.38 .35 .32 .18 .07
0
.03
0 0 0
4 6
.16 .11
0 0
8
.16
0
12 24 48 72
.12 0
0 0
0
0
0
0
0
0
0
0
96
0
0
0
0
120
0
0
0
0
1 2
Journal of Dairy Science Vol. 59, No. 2
0 0 0
Additionally, Table 1 shows average concentrations in plasma and milk of oxytetracycline in six cows after intrauterine administration of a chelated form at 4 mg/kg body weight. Four of the cows showed oxytetracycline activity in plasma. Peak concentration was lower in plasma of cows given chelated oxytetracycline than in those given nonchelated drug. Oxytetracycline activity appeared in 2-h and remained until the 12-h collection. Peak activity appeared at the 4-h collection. Two cows did not show any oxytetracycline in plasma. This may have been due to much of the drug being excreted via the cervix shortly after intrauterine treatment. No oxytetracycline activity appeared in the rrfilk of any of the cows after intrauterine administration of chelated oxytetracycline at 4 mg/kg body weight. Therefore, the chelated form may be the drug of choice for treating uterine infections in lactating cows. These average oxytetracycline concentrations (Table 1) suggest that milk from cows ~reated with the nonchelated form should be withheld at least 24 h while the milk from animals receiving the chelated form would not require withholding. The nonchelated form of oxytetracycline appeared in the milk at 4-h and remained through 24-h. This did not agree with Kendrick and Pier (9) who showed that there was no oxytetracycline in milk at 12, 24, 48, and 72-h posttreatment. They, however, used the rapid disc assay for penicillin activity (1) to determine the amount of oxytetracycline in the milk, an incorrect method. The chelated form of oxytetracycline crossed the uterine membrane into plasma and milk less completely than did the nonchelated form. This may have been due to the chelation of the oxytetracycline to the metal halides in the drug mixture. Therefore, this drug complex moved across the uterine membrane more slowly. REFERENCES
.04 .09 .14 .14 .10
1 Arret, B., and A. Kirschbaum. 1959. A rapid disc assay method for detecting penicillin in milk. J. Milk Food Technol. 329. 2 Bierschwal, C. J., and A. W. Uren. 1955. The absorption of sulfamethazine by the bovine uterus. J. Amer. Vet. Med. Ass. 126:398. 3 Bierschwal, C. J., and A. W. Uren. 1956. The absorption of chlortetracycline (Aureomycin) by the bovine uterus. J. Amer. Vet. Med. Ass. 129:373.
TECHNICAL NOTE 4 Bratton, A. C., and E. K. MarshaU. 1939. A new coupling c o m p o n e n t for sulfanilamide determination. J. Biol. Chem. 128:537. 5 Edman, L., O. Holmberg, I. Settergren, a n d C. B. Thorell. 1965. Resorption o f iodine in Lugol's solution and in an iodophos from the u t e r u s of cows. Nord. Vet. Med. 17:391. 6 Food a n d Drug Administration. 1968. Antibiotic residues in milk, dairy p r o d u c t s a n d animal tissues: Methods, reports and protocols. Guideline pamphlet on antibiotic analysis. 7 Huang, J., D. Hawkinson, and G. E. Miller. 1972. Passage o f Sulfadimethoxine from the bovine uterus into blood, milk, and urine. J. Dairy Sci. 55:705. (Abstr.) 8 Huang, J., a n d G. E. Miller. 1972. Mechanism of m o v e m e n t of sulfanilamide, sulfaethoxypyridazine, sulfamethazine, sulfadimethoxine, sulfacetamide, and salicylic acid across t h e uterine m e m b r a n e of the guinea pig. J. Dairy Sci. 55:705. (Abstr.) 9 Kendrick, J. W., and A. C. Pier. 1960. Antibiotic
10
11
12
13
14
317
levels in milk following intrauterine infusion. The California Vet., M a y - J u n e . Miller, G. E. 1971. Passage of drugs across the bovine uterine m e m b r a n e . Vet. Med. Rep., Agr. Ext. Serv. No. 4, 41, June. Miller, G. E., N. C. Banerjee, and C. M. Stowe, Jr. 1967. Drug m o v e m e n t between bovine milk and plasma as affected by milk pH. J. Dairy Sci. 50:1395. Miller, G. E., G. P. Bergt, W. M. Rose, D. B. Brunson, and R. G. Messer. 1973. Distribution of oxytetracycline in bovine plasma, milk, a n d urine following its intrauterine administration in two forms. J. Dairy Sci. 56:659. (Abstr.) Miller, G. E., and G. Rouse. 1970. Passage of drugs from the bovine uterus into blood, milk and urine. J. Dairy Sci. 53:652. (Abstr.) Miller, G. E., G. Rouse, a n d M. L. Fahning. 1971. Mechanism o f m o v e m e n t of sulfamethazine and sulfacetamide across the bovine uterine m e m b r a n e . J. Dairy Sci. 54:795. (Abstr.)
Journal of Dairy Science Vol. 59, No. 2
