Prostaglandin in Milk, Days Open, and Estrus Detection in Dairy Cows Treated with Prostaglandin F2 1 K. R. SIMMONS, S. C. MOSES, and B. L. PERKINS
Department of Animal Sciences University of Vermont Burlington 05405 ABSTRACT
Prostaglandin F2~ was used to induce estrus in postpartum dairy cows to decrease calving interval and time spent on heat checks. The amount of prostaglandin residue in milk after treatment also was investigated. Twenty-two control cows, checked for heat twice daily, were bred at estrus after 55 days postpartum. Sixty-three cows checked on days 55 to 60 were either bred if in estrus or treated with 30 mg of prostaglandin F2~ injected intramuscularly on day 60 and bred at induced estrus or at 75 h posttreatment. Twelve cows were in heat between days 55 and 60 and were bred. Twentynine of 51 treated cows were seen in estrus; all of these were bred and 51.7% conceived at this time. Treated cows had fewer days to first service than did controls, but days open were not different, 105.7 versus 101.8. Fe[tility was similar. Controls required more heat checks than did treated cows (39 to 8.2). Milk from 17 cows at two milkings before and ten milkings after treatment had similar concentrations of prostaglandin (698 + 27 pg/ml) except for the first posttreatment milking when prostaglandin concentration almost doubled (1,293 -+ 143 pg/ml). INTRODUCTION
Prostaglandin F2~ (PGFz~) has proved useful as a luteolytic agent for synchronizing estrus primarily in beef cows (7, 11, 13, 28, 30) and in dairy (2, 8, 17, 26) and beef heifers (5, 10, 11, 22). Analogues of PGF2~ have also
Received November 27, 1978. J Vermont Agricultural Experiment Station Journal Article No. 412. 1979 J Dairy Science 62:1443-1448
been used (4, 14, 23, 25, 29, 31). Trials with lactating dairy cows have been fewer (1, 15, 16, 20, 21, 29). Synchronizing large groups of dairy cows into estrus is probably less desirable than inducing estrus in a few females at a time. Regulation of estrus would be useful in reducing days open, in reducing the time spent on checks for estrus, and in scheduling breeding. Inducing ovulation at a predetermined time, without checks for estrus, is the ultimate goal. Under farm conditions, many cows are not noticed in estrus until some time after day 60 postpartum, thus delaying the time of first breeding and subsequent conception (1, 12, 19, 27). If all cows were induced to estrus and mated by days 60 to 65, then the number of days open probably would be reduced if fertility remained normal. The object of this investigation was to determine whether days open and time spent on checks for estrus would be reduced by using PGF2~ to induce estrus in lactating dairy cows at a predetermined time. If estrus-induction schemes are to be used widely in dairy herds, they should be integrated with existing husbandry practices and not require extensive changes in established procedures. Our investigation was planned with this thought. A further objective was to determine how prostaglandin in milk would be affected by treatment; little information is available. Two reports have indicated that prostaglandin in milk is elevated for one milking (18) or several milkings (9) after treatment. MATERIALS AND METHODS
All cows in the university herd were used over 1 yr. Four breeds and two housing systems were involved. Thirty-one cows (10 Ayrshires, 21 Holsteins) were in a free stall barn and 54 (7 Ayrshires, 4 Guernseys, 25 Holsteins, 18 Jerseys) were housed in a tie barn with comfort stalls. The cows ranged in age from 2 yr 8 mo to 8 yr 10 mo. The milking intervals were 11
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SIMMONS ET AL.
and 13 h. All cows were turned out daily at 0900 h for checks for estrus and for exercise while the barns were cleaned. Standing to be mounted was the only criterion for estrus, and breeding occurred no earlier than day 55 postpartum. Cows were assigned to treatment or control groups on calving date with every third cow placed in the control group. Treatment consisted of 30 mg PGFza-tham salt 2 injected im on the morning of day 60 postpartum. To avoid the first 4 days of the estrous cycle when P G F 2 a is ineffective as a luteolytic agent, twice daily estrous checks of cows in the treatment group began on day 55. Any cows showing estrus between days 55 to 60 were bred at that time. Those not showing estrus were treated on day 60 and then bred during the induced estrus. Treated cows not induced to estrus were inseminated at 75 h posttreatment although estrous checks were continued for 2 more days. Cows were treated with PGF2~ only once. Cows in the treatment group were checked every morning when the herd was turned out, but the late afternoon checks were not begun until day 55. Control cows were checked twice daily beginning on day 25 postpartum although breeding did not begin until day 55. All cows were rechecked twice for estrus from day 16 to 25 postbreeding and were rebred if in estrus. Pregnancy was determined by rectal palpation at 35 to 40 days postbreeding. One inseminator was used throughout the study. Seventeen cows receiving prostaglandin were used to monitor the effects of treatment on prostaglandin in milk. Representative aliquots were taken from the milk at each of two milkings before treatment and for 10 milkings after treatment. Milk samples were stored at - 2 0 C until they were assayed for prostaglandin. In the same 17 cows blood samples were taken once per day for 6 days beginning on the morning of treatment preceding PGF2a. All other treated cows had blood drawn once prior to treatment and also before insemination at 75 h if estrus was not induced. Blood samples were collected in tubes with sodium citrate as an anticoagulant. The plasma was separated off
2Prostin F2Alpha , Upjohn Co., Kalamazoo, MI courtesy of J. W. Lauderdale. Journal of Dairy Science Vol. 62, No. 9, 1979
and stored at - 2 0 C until it was assayed for progesterone. The presence or absence of corpora lutea (CL), detected by rectal palpation, was noted for all cows receiving PGF2a. Normally cows without a CL would not be treated with PGF2 aHowever, we did not use the presence or absence of a CL as a criterion for treatment because most farmers would not palpate the ovaries, and we attempted to mimic the use of PGF2a by the average farmer. The ovaries were palpated again at 72 to 75 h post-PGF2a for those cows not induced to estrus. Because of Food and Drug Administration (FDA) rulings, we were obligated to increase the discard period for milk from 2 to 10 milkings after treatment. This change occurred about halfway through the experiment. Prostaglandin in milk was determined by a double antibody, radioimmunoassay (RIA) procedure. All samples from a particular cow were in one assay. To extract prostaglandins from milk, we used a method similar to the one described by English and Williams (6). Five milliliters of milk were extracted with 5 ml of acidified ethyl alcohol (3 N HC1 to pH 4) and twice with 3 ml of ethyl acetate. The supernatant was stored in a freezer overnight and then filtered through Whatman #1 filter paper by vacuum and an additional rinse of 3 ml of ethyl acetate. The filtrate was condensed at 45 C under nitrogen to an approximate volume of 3 ml. Methyl alcohol and hexane (2 ml each) were mixed with the filtrate, and the tubes were centrifuged. The hexane layer was pipetted off and discarded. The remaining solution was evaporated to slightly less than 2 ml and adjusted to a final volume of 2 ml with tris buffer. The extracted samples were stored at 4 C overnight until they were assayed. Extraction efficiency, as determined by added tritiated prostaglandin, was 90 to 98%. Prostaglandin F2a, thus extracted from milk, was quantified with a commercial radioimmunoassay kit (Clinical Assays, Inc., Cambridge, MA). Sensitivity of the assay was 10 pg. Recovery experiments were by assaying known amounts of PGF2a standard added to a pooled milk sample. The amounts added were 600, 1200, 2400, and 3600 pg/ml, and those recovered were 620, 1270, 2420, and 4570 pg/ml after correcting for endogenous amounts and efficiency of extraction. The regression equation
PROSTAGLANDIN IN MILK AFTER TREATMENT predicting the amount recovered from the amount added is ~ = 220 + 1.31 (X-195). The r z was .98. Intra- and interassay coefficients of variation were 4.3% and 10.6%• Crossreactivity data, supplied by Clinical Assays, Inc., indicated that the antibody crossreacts with P G F I ~ to 42% but to less than .02% with PGE, PGA, or PGB. Progesterone in plasma was quantified by a double antibody radioimmunoassay procedure kit prepared by Micromedic Systems, Horsham, PA. Recovery experiments for progesterone added to pooled plasma at .265, 1.04, 4.16, and 16.66 ng/ml yielded .2, .95, 3.12, and 12.62 ng/ml after corrections for extraction efficiency and endogenous progesterone. The regression equation describing this relationship is ~ = 4.22 + .75 (X-5.53). The r 2 was .98. The intra- and interassay coefficients of variation were 7.9% and 12.6%. Crossreactivity of the antibody supplied with this kit is 42% with 5-pregnan-3,20-dione, 17% and 16% with 11a and/3 hydroxyprogesterone, and less than 2.4% with other physiologically important steroids. The sensitivity of this assay is .03 ng/ml. All samples from a particular cow were included in the same assay.
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number expected to be in estrus during 5 days. Of the 51 cows actually treated, 29 (56.9%) were detected in estrus during the 5 days after treatment (Table 1). These cows were designated as responders, and those not detected in estrus during this time were designated as nonresponders. Fifteen (51.7%) of the 29 responders conceived to the induced estrus. Of the 22 nonresponders, three conceived to breeding at 75 h posttreatment. The number of services per conception for the treatment group as a whole was not significantly different (P
Department of Animal Sciences University of Vermont Burlington 05405 ABSTRACT
Prostaglandin F2~ was used to induce estrus in postpartum dairy cows to decrease calving interval and time spent on heat checks. The amount of prostaglandin residue in milk after treatment also was investigated. Twenty-two control cows, checked for heat twice daily, were bred at estrus after 55 days postpartum. Sixty-three cows checked on days 55 to 60 were either bred if in estrus or treated with 30 mg of prostaglandin F2~ injected intramuscularly on day 60 and bred at induced estrus or at 75 h posttreatment. Twelve cows were in heat between days 55 and 60 and were bred. Twentynine of 51 treated cows were seen in estrus; all of these were bred and 51.7% conceived at this time. Treated cows had fewer days to first service than did controls, but days open were not different, 105.7 versus 101.8. Fe[tility was similar. Controls required more heat checks than did treated cows (39 to 8.2). Milk from 17 cows at two milkings before and ten milkings after treatment had similar concentrations of prostaglandin (698 + 27 pg/ml) except for the first posttreatment milking when prostaglandin concentration almost doubled (1,293 -+ 143 pg/ml). INTRODUCTION
Prostaglandin F2~ (PGFz~) has proved useful as a luteolytic agent for synchronizing estrus primarily in beef cows (7, 11, 13, 28, 30) and in dairy (2, 8, 17, 26) and beef heifers (5, 10, 11, 22). Analogues of PGF2~ have also
Received November 27, 1978. J Vermont Agricultural Experiment Station Journal Article No. 412. 1979 J Dairy Science 62:1443-1448
been used (4, 14, 23, 25, 29, 31). Trials with lactating dairy cows have been fewer (1, 15, 16, 20, 21, 29). Synchronizing large groups of dairy cows into estrus is probably less desirable than inducing estrus in a few females at a time. Regulation of estrus would be useful in reducing days open, in reducing the time spent on checks for estrus, and in scheduling breeding. Inducing ovulation at a predetermined time, without checks for estrus, is the ultimate goal. Under farm conditions, many cows are not noticed in estrus until some time after day 60 postpartum, thus delaying the time of first breeding and subsequent conception (1, 12, 19, 27). If all cows were induced to estrus and mated by days 60 to 65, then the number of days open probably would be reduced if fertility remained normal. The object of this investigation was to determine whether days open and time spent on checks for estrus would be reduced by using PGF2~ to induce estrus in lactating dairy cows at a predetermined time. If estrus-induction schemes are to be used widely in dairy herds, they should be integrated with existing husbandry practices and not require extensive changes in established procedures. Our investigation was planned with this thought. A further objective was to determine how prostaglandin in milk would be affected by treatment; little information is available. Two reports have indicated that prostaglandin in milk is elevated for one milking (18) or several milkings (9) after treatment. MATERIALS AND METHODS
All cows in the university herd were used over 1 yr. Four breeds and two housing systems were involved. Thirty-one cows (10 Ayrshires, 21 Holsteins) were in a free stall barn and 54 (7 Ayrshires, 4 Guernseys, 25 Holsteins, 18 Jerseys) were housed in a tie barn with comfort stalls. The cows ranged in age from 2 yr 8 mo to 8 yr 10 mo. The milking intervals were 11
1443
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SIMMONS ET AL.
and 13 h. All cows were turned out daily at 0900 h for checks for estrus and for exercise while the barns were cleaned. Standing to be mounted was the only criterion for estrus, and breeding occurred no earlier than day 55 postpartum. Cows were assigned to treatment or control groups on calving date with every third cow placed in the control group. Treatment consisted of 30 mg PGFza-tham salt 2 injected im on the morning of day 60 postpartum. To avoid the first 4 days of the estrous cycle when P G F 2 a is ineffective as a luteolytic agent, twice daily estrous checks of cows in the treatment group began on day 55. Any cows showing estrus between days 55 to 60 were bred at that time. Those not showing estrus were treated on day 60 and then bred during the induced estrus. Treated cows not induced to estrus were inseminated at 75 h posttreatment although estrous checks were continued for 2 more days. Cows were treated with PGF2~ only once. Cows in the treatment group were checked every morning when the herd was turned out, but the late afternoon checks were not begun until day 55. Control cows were checked twice daily beginning on day 25 postpartum although breeding did not begin until day 55. All cows were rechecked twice for estrus from day 16 to 25 postbreeding and were rebred if in estrus. Pregnancy was determined by rectal palpation at 35 to 40 days postbreeding. One inseminator was used throughout the study. Seventeen cows receiving prostaglandin were used to monitor the effects of treatment on prostaglandin in milk. Representative aliquots were taken from the milk at each of two milkings before treatment and for 10 milkings after treatment. Milk samples were stored at - 2 0 C until they were assayed for prostaglandin. In the same 17 cows blood samples were taken once per day for 6 days beginning on the morning of treatment preceding PGF2a. All other treated cows had blood drawn once prior to treatment and also before insemination at 75 h if estrus was not induced. Blood samples were collected in tubes with sodium citrate as an anticoagulant. The plasma was separated off
2Prostin F2Alpha , Upjohn Co., Kalamazoo, MI courtesy of J. W. Lauderdale. Journal of Dairy Science Vol. 62, No. 9, 1979
and stored at - 2 0 C until it was assayed for progesterone. The presence or absence of corpora lutea (CL), detected by rectal palpation, was noted for all cows receiving PGF2a. Normally cows without a CL would not be treated with PGF2 aHowever, we did not use the presence or absence of a CL as a criterion for treatment because most farmers would not palpate the ovaries, and we attempted to mimic the use of PGF2a by the average farmer. The ovaries were palpated again at 72 to 75 h post-PGF2a for those cows not induced to estrus. Because of Food and Drug Administration (FDA) rulings, we were obligated to increase the discard period for milk from 2 to 10 milkings after treatment. This change occurred about halfway through the experiment. Prostaglandin in milk was determined by a double antibody, radioimmunoassay (RIA) procedure. All samples from a particular cow were in one assay. To extract prostaglandins from milk, we used a method similar to the one described by English and Williams (6). Five milliliters of milk were extracted with 5 ml of acidified ethyl alcohol (3 N HC1 to pH 4) and twice with 3 ml of ethyl acetate. The supernatant was stored in a freezer overnight and then filtered through Whatman #1 filter paper by vacuum and an additional rinse of 3 ml of ethyl acetate. The filtrate was condensed at 45 C under nitrogen to an approximate volume of 3 ml. Methyl alcohol and hexane (2 ml each) were mixed with the filtrate, and the tubes were centrifuged. The hexane layer was pipetted off and discarded. The remaining solution was evaporated to slightly less than 2 ml and adjusted to a final volume of 2 ml with tris buffer. The extracted samples were stored at 4 C overnight until they were assayed. Extraction efficiency, as determined by added tritiated prostaglandin, was 90 to 98%. Prostaglandin F2a, thus extracted from milk, was quantified with a commercial radioimmunoassay kit (Clinical Assays, Inc., Cambridge, MA). Sensitivity of the assay was 10 pg. Recovery experiments were by assaying known amounts of PGF2a standard added to a pooled milk sample. The amounts added were 600, 1200, 2400, and 3600 pg/ml, and those recovered were 620, 1270, 2420, and 4570 pg/ml after correcting for endogenous amounts and efficiency of extraction. The regression equation
PROSTAGLANDIN IN MILK AFTER TREATMENT predicting the amount recovered from the amount added is ~ = 220 + 1.31 (X-195). The r z was .98. Intra- and interassay coefficients of variation were 4.3% and 10.6%• Crossreactivity data, supplied by Clinical Assays, Inc., indicated that the antibody crossreacts with P G F I ~ to 42% but to less than .02% with PGE, PGA, or PGB. Progesterone in plasma was quantified by a double antibody radioimmunoassay procedure kit prepared by Micromedic Systems, Horsham, PA. Recovery experiments for progesterone added to pooled plasma at .265, 1.04, 4.16, and 16.66 ng/ml yielded .2, .95, 3.12, and 12.62 ng/ml after corrections for extraction efficiency and endogenous progesterone. The regression equation describing this relationship is ~ = 4.22 + .75 (X-5.53). The r 2 was .98. The intra- and interassay coefficients of variation were 7.9% and 12.6%. Crossreactivity of the antibody supplied with this kit is 42% with 5-pregnan-3,20-dione, 17% and 16% with 11a and/3 hydroxyprogesterone, and less than 2.4% with other physiologically important steroids. The sensitivity of this assay is .03 ng/ml. All samples from a particular cow were included in the same assay.
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number expected to be in estrus during 5 days. Of the 51 cows actually treated, 29 (56.9%) were detected in estrus during the 5 days after treatment (Table 1). These cows were designated as responders, and those not detected in estrus during this time were designated as nonresponders. Fifteen (51.7%) of the 29 responders conceived to the induced estrus. Of the 22 nonresponders, three conceived to breeding at 75 h posttreatment. The number of services per conception for the treatment group as a whole was not significantly different (P
