Hormonal Induction of Lactation: Estrogen and Progesterone in Milk I R. N A R E N D R A N , R. R. H A C K E R , V. G. S M I T H 2, and A. LUN Department of Animal and Poultry Science University of Guelph Guelph, Ontario, Canada N1G 2W1 ABSTRACT
estradiol-17~ and progesterone for 7 days. Narendran et al. (18) reported that the milk secreted following this induction procedure did not differ from that after normal parturition and that histological sections of mammary glands in the induced cows did not show abnormalities. Erb et al (5) measured progesterone and total free estrogen in peripheral plasma and urinary estrogens in nonpregnant nonlactating cows treated with estradiol-17~ and progesterone for 7 days and concluded that the subcutaneous injections of the hormones probably slowed absorption and delayed excretion of the hormones. Our aim was to study concentrations of estrogen and progesterone in milk of cows and heifers successfully induced to lactate with estradiol-17j3 and progesterone therapy and to compare these with concentrations in postpartum milk at a corresponding stage of lactation.
Estrogen and progesterone in milk during the first 21 days of induced and postpartum lactation in Holstein cows and heifers were estimated by assay procedures. Lactation was induced with estradiol-17/3 and progesterone treatment for 7 days. Estrogen and progesterone in induced lactations differed from concentrations in postpartum lactations. In early lactation estrogen was higher in postpartum milk (521 -+ 103 pg/ml on day 1) than in induced milk (336 + 46 pg/ml on day 1), but after day 7 the reverse was true (192 _+ 33 pg/ml and 233 -+ 32 pg/ml on day 7). Progesterone remained higher in induced lactation through the first 21 days than in postpartum lactation with the exception of day 19. Progesterone in postpartum milk increased from 4 -+ 1 ng/ml on day 1 to 11 -+ 2 ng/ml on day 21. Progesterone in induced milk showed greater fluctuation (11 -+ 3 ng/ml on day 1 and 22 + 9 ng/mi on day 3) but gradually decreased to 12 _+ 2 ng/ml on day 21 (11 ± 2 ng/ml on day 21 of postpartum lactation).
MATERIALS AND METHODS Animals
All animals were Holstein and were housed in a tie-stall barn. They received a ration of haylage plus grain mixture. All animals were nonpregnant and nonlactating.
INTRODUCTION
Induced lactation in the diary cow has received fresh impetus in recent years. Smith et al. (25) and Smith and Schanbacher (26, 27) reported successful induction of lactation in nonlactating cows and heifers by a procedure involving twice daily subcutaneous injections of
Received July 5, 1978. 1 Research supported by the Ontario Ministry of Agriculture and Food and the National Research Council of Canada. Grant No. A-6247. 2Upjohn Company, Unit 9602-25-5, Kalamazoo, MI 49001. 1979
J Dairy Sci 62:1069-1075
Hormones and Injections
We injected subcutaneously 1, 3, 5 ( 1 0 ) estratrien - 3, 17/3 diol (estradiol-17~) and 4 pregnene - 3, 20 dione (progesterone) (Sigma Chemical Co., St. Louis, MO) dissolved in absolute alcohol at dosages of .1 mg/kg and .25 mg/kg bodyweight, respectively, for 7 days as in (18). Milk Samples
Milk samples (500 ml) were obtained at morning milking from 18 normally calved cows and heifers for 21 days beginning on day 1 of
1069
1070
NARENDRAN ET AL.
lactation. Milk samples from 18 cows and heifers successfully induced to lactate (18) following estradiol-progesterone therapy were collected similarly during the first 21 days of induced lactation. All milk samples were collected at the end of milking from the weigh jar into polyethylene sampling bags and beakers and were stored at - 1 8 C. Milk samples to be assayed were sorted the previous evening and allowed to thaw overnight at 4 C. The thawed milk samples were homogenized for 3 rain by an ultrasonic device (Biosonik III, Bronwell Scientific, Rochester, NY) at maximum intensity prior to being used for fat testing and hormone assays. Milk Fat Analysis
All milk samples assayed for hormones were analyzed in duplicate for fat at the central milk testing laboratories, Guelph, Ontario. The samples for fat analysis were preserved with "Lactabs" (mercuric chloride plus potassium dichromate) (Thompson and Capper Ltd., Liverpool, U.K.) during post-thawing storage at 4 C prior to analysis. Extraction of Estrogen and Progesterone from Milk
Milk samples in .5-ml quantities were pipetted in duplicate into glass extraction t u b e s and were extracted twice with 6 ml and 4 ml of ethyl ether for 2 rain and 1 min for each extraction, respectively. Following each extraction the tubes were immersed for 5 min in a methanol bath (Hotpack, Waterloo, Ontario) maintained at - 2 0 C, the supernatant decanted into glass culture tubes and dried under nitrogen in a 40 C waterbath. The dry extract was dissolved in 4 ml of methanol: water (7:3), vortexed for 15 s, and incubated in a 40 C waterbath for 1 h. The excess fat precipitated by this step was compacted by centrifugation at 1000 x g for 10 rain at - 1 0 C. The tubes were placed in a methanol bath at - 2 0 C for 15 rain prior to supernatant being partitioned for use in the assays of estrogen and progesterone. Competitive Protein Binding (CPB) Assay for Progesterone
Subsequent to the fat precipitation step, 2 ml of the methanol extract were pipetted into glass extraction tubes. Four milliliters of double Journal of Dairy Science Vol. 62, No. 7, 1979
distilled water and 8 ml of petroleum ether (Fraction between 38 and 42 C ) t h e n were added and the contents vortexed for 1 min (12). The tubes were immersed in a dry ice acetone bath for 2 rain to facilitate accurate separation of the petroleum ether phase from the methanol phase, which freezes. The supernatant was decanted into glass culture tubes and dried under nitrogen in 40 C waterbath. No fat globules were observed in the ,glass culture tubes post-drying. The assay was according to the method of Robertson and Sarda (21). The source of corticosterone binding globulin was adult male dog plasma. For the standard curve, progesterone at concentrations of 0, .5, 1, 2, 4, 6, 8, and 10 ng in duplicate was used. The standard tubes also were dried under nitrogen in a 40 C waterbath. The CPB assay for progesterone subsequent to petroleum ether extraction has been specific for progesterone (4, 12, 13, 21). Radioimmunoassay (RIA) for Estrogen
Subsequent to the fat precipitation step, 1 ml of the methanol extract was pipetted into glass culture tubes and evaporated to dryness under nitrogen in a 40 C waterbath. No fat globules were observed in the glass culture tubes post-drying. The assay was according to the method of Britt, Kittok, and Harrison (2). Antisera specific to estrogen were obtained from R. D. Randel, Texas A&M University, Overton, TX. Cross reactivity of these antisera has been reported (5). For the standard curve, estradiol-17J3 at concentrations of 0, 2, 5, 10, 20, 50, 100, and 200 pg, in duplicate was used. The standard tubes also were dried under nitrogen in 40 C waterbath. Two hundred microliters of 1:60,000 estrogen specific antisera in gelatine phosphate buffer (GPB:.IM phosphate buffer pH 7, containing .15M sodium chloride .015M sodium azide and .1% gelatine) were used in each assay tube. Assessment of Blank Values and Recovery Percentages
Double distilled watec was used to evaluate the method blank in the competitive protein binding and radioimmunoassays for milk progesterone and estrogen, respectively. Double distilled water (.5 ml) was incorporated in duplicate with each set of milk assays and was
ESTROGEN AND PROGESTERONE IN MILK
1071
corrections were made to account for the blank estimates of estrogens and progesterone. Amounts of estrogen and progesterone in various volumes of the identical milk sample were compared to those of their respective standard curves for parallelism by F-test. The observed estrogen and progesterone in milk during induced and postpartum lactations were regressed on fat percentage, and the regression coefficients were used to estimate estrogen and progesterone for each fat percent. The differences between observed and estimated estrogen and progesterone were used to compare by multiple profile analysis (9) the hormone in milk during induced and postpartum lactation. Students' t-test (29) was used to test the significance of the differences in hormone concentrations on specific days between induced and postpartum lactations. Correlation coefficients between fat percentages and estrogen and progesterone in milk were determined for induced and postpartum lactations.
subject to the same procedures as the milk samples. When the blanks represented more than 10% of the usable portion of the standard curve, the assay was arbitrarily invalid as recommended by Abraham (1). Recovery of the estrogens and progesterone by the extraction procedure was estimated by adding 1500 cpm of [3H]estradiol-1713 and 12,O00 cpm of [3H]progesterone in 10 ~tl ethanol t o . 5 - m l aliquots of milk and equilibrating for 30 min prior to subjecting the milk to the extraction procedures. Determinations were separate and duplicate for estrogens and progesterone for each group of assays. The petroleum ether and methanol extract in volumes used in the corresponding progesterone and estrogen assays proper were measured into scintillation vials and dried prior to quantification. Validation of Assays
To validate the estrogen and progesterone assays, recovery of added estradiol-1713 and progesterone to milk was determined, and parallelism between CPB and RIA displacement curves for progesterone and estrogens, respectively, in various volumes of a given milk sample (.2 to 1 ml for progesterone and .02 to 1 ml for estrogens) and their corresponding standard curves was demonstrated.
RESULTS A N D DISCUSSION Validation of Assay Procedures, Recovery, and Blank Estimates
Displacement curves prepared by assaying increasing amounts of standard cold estradiol1713 and progesterone and extracts of increasing volumes of milk were parallel. The slopes of curves for estrogen and progesterone in various volumes of milk and their respective standard curves were not significantly different. When
Statistical Analysis
Estrogen and progesterone of milk in the assays were corrected for procedural losses. No
TABLE 1. Recovery of known quantities of estradiol-17/5 and progesterone from milk. Amount added
Amount recovereda --
X
--
--SE
Recovery
--
Estradiol-17/3 (pg) 25 50 100 200
31.53 55.16 98.66 191.55
.67 4.48 7.81 14.04
126.12 110.32 98.66 95.77
2.46 6.78 8.36
.11 .43 .41
123.00 113.00 104.50
Progesterone (ng) 2 6 8 a
.
.
.
Stx determinations (adjusted for procedural losses). Journal of Dairy Science Vol. 62, No. 7, 1979
1072
NARENDRAN ET AL.
o.----~
o
POST PARTII'-I
o L~cc~
,d
DAYSOF LACTATI~
Figure 1. Estrogen in milk during the first 21 days of induced and postpartum lactations (n=18 per group).
25, 50, 100, and 200 pg, respectively, of estradiol-17/3 and 2, 6, and 8 ng, respectively, of progesterone were added to milk, they were recovered by assay (Table 1). Mean recoveries of [3H]estradiol-1713 and [3H] progesterone added to milk were 88 -+ 1% (SE) and 79 -+ .8% (SE). When double distilled water was handled as a sample, the mean of the inhibiting activity (Method blank) was 8.6 + .7 pg and .38 + .04 ng for estrogens and progesterone. Estrogen in Induced and Postpartum Milk
The mean estrogen in milk during the first 3 wk of induced and postpartum lactation are in Figure 1. In the first 21 days of induced lactation estrogen declined from 334 + 46 pg/ml on day 1 to 202 + 30 pg/ml on day 19. In the postpartum period, estrogen declined from 521 + 103 pg/ml on day 1 to 170 -+ 24 pg/ml on day 9 and increased again starting on day 15 to plateau at 211 + 34 pg/ml on day 19. In the immediate postpartum period estrogen in milk was higher than in induced milk, but after day 6 the pattern changed with induced milk showing more than postpartum milk. The changes in estrogen in induced and postpartum Journal of Dairy Science Vol. 62, No. 7, 1979
milk paralleled each other after day 6 of lactation. Total free estrogen in milk probably was underestimated because estradiol-17t3 standard was used for quantification and the rate of displacement of [3 H] estradiol-17t3 by standard estradiol-17ce or estrone was .5 that of standard estradiol-1713 at 50% displacement of [3H] estradiol-17~. Estrogen in milk during induced and postpartum lactation as compared by multiple profile analysis demonstrated parallelism (P
estradiol-17~ and progesterone for 7 days. Narendran et al. (18) reported that the milk secreted following this induction procedure did not differ from that after normal parturition and that histological sections of mammary glands in the induced cows did not show abnormalities. Erb et al (5) measured progesterone and total free estrogen in peripheral plasma and urinary estrogens in nonpregnant nonlactating cows treated with estradiol-17~ and progesterone for 7 days and concluded that the subcutaneous injections of the hormones probably slowed absorption and delayed excretion of the hormones. Our aim was to study concentrations of estrogen and progesterone in milk of cows and heifers successfully induced to lactate with estradiol-17j3 and progesterone therapy and to compare these with concentrations in postpartum milk at a corresponding stage of lactation.
Estrogen and progesterone in milk during the first 21 days of induced and postpartum lactation in Holstein cows and heifers were estimated by assay procedures. Lactation was induced with estradiol-17/3 and progesterone treatment for 7 days. Estrogen and progesterone in induced lactations differed from concentrations in postpartum lactations. In early lactation estrogen was higher in postpartum milk (521 -+ 103 pg/ml on day 1) than in induced milk (336 + 46 pg/ml on day 1), but after day 7 the reverse was true (192 _+ 33 pg/ml and 233 -+ 32 pg/ml on day 7). Progesterone remained higher in induced lactation through the first 21 days than in postpartum lactation with the exception of day 19. Progesterone in postpartum milk increased from 4 -+ 1 ng/ml on day 1 to 11 -+ 2 ng/ml on day 21. Progesterone in induced milk showed greater fluctuation (11 -+ 3 ng/ml on day 1 and 22 + 9 ng/mi on day 3) but gradually decreased to 12 _+ 2 ng/ml on day 21 (11 ± 2 ng/ml on day 21 of postpartum lactation).
MATERIALS AND METHODS Animals
All animals were Holstein and were housed in a tie-stall barn. They received a ration of haylage plus grain mixture. All animals were nonpregnant and nonlactating.
INTRODUCTION
Induced lactation in the diary cow has received fresh impetus in recent years. Smith et al. (25) and Smith and Schanbacher (26, 27) reported successful induction of lactation in nonlactating cows and heifers by a procedure involving twice daily subcutaneous injections of
Received July 5, 1978. 1 Research supported by the Ontario Ministry of Agriculture and Food and the National Research Council of Canada. Grant No. A-6247. 2Upjohn Company, Unit 9602-25-5, Kalamazoo, MI 49001. 1979
J Dairy Sci 62:1069-1075
Hormones and Injections
We injected subcutaneously 1, 3, 5 ( 1 0 ) estratrien - 3, 17/3 diol (estradiol-17~) and 4 pregnene - 3, 20 dione (progesterone) (Sigma Chemical Co., St. Louis, MO) dissolved in absolute alcohol at dosages of .1 mg/kg and .25 mg/kg bodyweight, respectively, for 7 days as in (18). Milk Samples
Milk samples (500 ml) were obtained at morning milking from 18 normally calved cows and heifers for 21 days beginning on day 1 of
1069
1070
NARENDRAN ET AL.
lactation. Milk samples from 18 cows and heifers successfully induced to lactate (18) following estradiol-progesterone therapy were collected similarly during the first 21 days of induced lactation. All milk samples were collected at the end of milking from the weigh jar into polyethylene sampling bags and beakers and were stored at - 1 8 C. Milk samples to be assayed were sorted the previous evening and allowed to thaw overnight at 4 C. The thawed milk samples were homogenized for 3 rain by an ultrasonic device (Biosonik III, Bronwell Scientific, Rochester, NY) at maximum intensity prior to being used for fat testing and hormone assays. Milk Fat Analysis
All milk samples assayed for hormones were analyzed in duplicate for fat at the central milk testing laboratories, Guelph, Ontario. The samples for fat analysis were preserved with "Lactabs" (mercuric chloride plus potassium dichromate) (Thompson and Capper Ltd., Liverpool, U.K.) during post-thawing storage at 4 C prior to analysis. Extraction of Estrogen and Progesterone from Milk
Milk samples in .5-ml quantities were pipetted in duplicate into glass extraction t u b e s and were extracted twice with 6 ml and 4 ml of ethyl ether for 2 rain and 1 min for each extraction, respectively. Following each extraction the tubes were immersed for 5 min in a methanol bath (Hotpack, Waterloo, Ontario) maintained at - 2 0 C, the supernatant decanted into glass culture tubes and dried under nitrogen in a 40 C waterbath. The dry extract was dissolved in 4 ml of methanol: water (7:3), vortexed for 15 s, and incubated in a 40 C waterbath for 1 h. The excess fat precipitated by this step was compacted by centrifugation at 1000 x g for 10 rain at - 1 0 C. The tubes were placed in a methanol bath at - 2 0 C for 15 rain prior to supernatant being partitioned for use in the assays of estrogen and progesterone. Competitive Protein Binding (CPB) Assay for Progesterone
Subsequent to the fat precipitation step, 2 ml of the methanol extract were pipetted into glass extraction tubes. Four milliliters of double Journal of Dairy Science Vol. 62, No. 7, 1979
distilled water and 8 ml of petroleum ether (Fraction between 38 and 42 C ) t h e n were added and the contents vortexed for 1 min (12). The tubes were immersed in a dry ice acetone bath for 2 rain to facilitate accurate separation of the petroleum ether phase from the methanol phase, which freezes. The supernatant was decanted into glass culture tubes and dried under nitrogen in 40 C waterbath. No fat globules were observed in the ,glass culture tubes post-drying. The assay was according to the method of Robertson and Sarda (21). The source of corticosterone binding globulin was adult male dog plasma. For the standard curve, progesterone at concentrations of 0, .5, 1, 2, 4, 6, 8, and 10 ng in duplicate was used. The standard tubes also were dried under nitrogen in a 40 C waterbath. The CPB assay for progesterone subsequent to petroleum ether extraction has been specific for progesterone (4, 12, 13, 21). Radioimmunoassay (RIA) for Estrogen
Subsequent to the fat precipitation step, 1 ml of the methanol extract was pipetted into glass culture tubes and evaporated to dryness under nitrogen in a 40 C waterbath. No fat globules were observed in the glass culture tubes post-drying. The assay was according to the method of Britt, Kittok, and Harrison (2). Antisera specific to estrogen were obtained from R. D. Randel, Texas A&M University, Overton, TX. Cross reactivity of these antisera has been reported (5). For the standard curve, estradiol-17J3 at concentrations of 0, 2, 5, 10, 20, 50, 100, and 200 pg, in duplicate was used. The standard tubes also were dried under nitrogen in 40 C waterbath. Two hundred microliters of 1:60,000 estrogen specific antisera in gelatine phosphate buffer (GPB:.IM phosphate buffer pH 7, containing .15M sodium chloride .015M sodium azide and .1% gelatine) were used in each assay tube. Assessment of Blank Values and Recovery Percentages
Double distilled watec was used to evaluate the method blank in the competitive protein binding and radioimmunoassays for milk progesterone and estrogen, respectively. Double distilled water (.5 ml) was incorporated in duplicate with each set of milk assays and was
ESTROGEN AND PROGESTERONE IN MILK
1071
corrections were made to account for the blank estimates of estrogens and progesterone. Amounts of estrogen and progesterone in various volumes of the identical milk sample were compared to those of their respective standard curves for parallelism by F-test. The observed estrogen and progesterone in milk during induced and postpartum lactations were regressed on fat percentage, and the regression coefficients were used to estimate estrogen and progesterone for each fat percent. The differences between observed and estimated estrogen and progesterone were used to compare by multiple profile analysis (9) the hormone in milk during induced and postpartum lactation. Students' t-test (29) was used to test the significance of the differences in hormone concentrations on specific days between induced and postpartum lactations. Correlation coefficients between fat percentages and estrogen and progesterone in milk were determined for induced and postpartum lactations.
subject to the same procedures as the milk samples. When the blanks represented more than 10% of the usable portion of the standard curve, the assay was arbitrarily invalid as recommended by Abraham (1). Recovery of the estrogens and progesterone by the extraction procedure was estimated by adding 1500 cpm of [3H]estradiol-1713 and 12,O00 cpm of [3H]progesterone in 10 ~tl ethanol t o . 5 - m l aliquots of milk and equilibrating for 30 min prior to subjecting the milk to the extraction procedures. Determinations were separate and duplicate for estrogens and progesterone for each group of assays. The petroleum ether and methanol extract in volumes used in the corresponding progesterone and estrogen assays proper were measured into scintillation vials and dried prior to quantification. Validation of Assays
To validate the estrogen and progesterone assays, recovery of added estradiol-1713 and progesterone to milk was determined, and parallelism between CPB and RIA displacement curves for progesterone and estrogens, respectively, in various volumes of a given milk sample (.2 to 1 ml for progesterone and .02 to 1 ml for estrogens) and their corresponding standard curves was demonstrated.
RESULTS A N D DISCUSSION Validation of Assay Procedures, Recovery, and Blank Estimates
Displacement curves prepared by assaying increasing amounts of standard cold estradiol1713 and progesterone and extracts of increasing volumes of milk were parallel. The slopes of curves for estrogen and progesterone in various volumes of milk and their respective standard curves were not significantly different. When
Statistical Analysis
Estrogen and progesterone of milk in the assays were corrected for procedural losses. No
TABLE 1. Recovery of known quantities of estradiol-17/5 and progesterone from milk. Amount added
Amount recovereda --
X
--
--SE
Recovery
--
Estradiol-17/3 (pg) 25 50 100 200
31.53 55.16 98.66 191.55
.67 4.48 7.81 14.04
126.12 110.32 98.66 95.77
2.46 6.78 8.36
.11 .43 .41
123.00 113.00 104.50
Progesterone (ng) 2 6 8 a
.
.
.
Stx determinations (adjusted for procedural losses). Journal of Dairy Science Vol. 62, No. 7, 1979
1072
NARENDRAN ET AL.
o.----~
o
POST PARTII'-I
o L~cc~
,d
DAYSOF LACTATI~
Figure 1. Estrogen in milk during the first 21 days of induced and postpartum lactations (n=18 per group).
25, 50, 100, and 200 pg, respectively, of estradiol-17/3 and 2, 6, and 8 ng, respectively, of progesterone were added to milk, they were recovered by assay (Table 1). Mean recoveries of [3H]estradiol-1713 and [3H] progesterone added to milk were 88 -+ 1% (SE) and 79 -+ .8% (SE). When double distilled water was handled as a sample, the mean of the inhibiting activity (Method blank) was 8.6 + .7 pg and .38 + .04 ng for estrogens and progesterone. Estrogen in Induced and Postpartum Milk
The mean estrogen in milk during the first 3 wk of induced and postpartum lactation are in Figure 1. In the first 21 days of induced lactation estrogen declined from 334 + 46 pg/ml on day 1 to 202 + 30 pg/ml on day 19. In the postpartum period, estrogen declined from 521 + 103 pg/ml on day 1 to 170 -+ 24 pg/ml on day 9 and increased again starting on day 15 to plateau at 211 + 34 pg/ml on day 19. In the immediate postpartum period estrogen in milk was higher than in induced milk, but after day 6 the pattern changed with induced milk showing more than postpartum milk. The changes in estrogen in induced and postpartum Journal of Dairy Science Vol. 62, No. 7, 1979
milk paralleled each other after day 6 of lactation. Total free estrogen in milk probably was underestimated because estradiol-17t3 standard was used for quantification and the rate of displacement of [3 H] estradiol-17t3 by standard estradiol-17ce or estrone was .5 that of standard estradiol-1713 at 50% displacement of [3H] estradiol-17~. Estrogen in milk during induced and postpartum lactation as compared by multiple profile analysis demonstrated parallelism (P
