Hormonal Induction of Lactation: Estrogen and
Progesterone in Milk I
ABSTRACT
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 treat-
ment for 7 days. Estrogen and progester-
one 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 post-
partum 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).
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
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
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 progester-
one and total free estrogen in peripheral plasma
and urinary estrogens in nonpregnant nonlactat-
ing cows treated with estradiol-17~ and proges-
terone 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 post-
partum milk at a corresponding stage of lacta-
tion.
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.
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 extrac-
tion 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 nitro-
gen 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 super-
natant 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 proges-
terone 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
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 equilibrat-
ing for 30 min prior to subjecting the milk to
the extraction procedures. Determinations were
separate and duplicate for estrogens and proges-
terone 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, respec-
tively, 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.
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 --
Estradiol-17/3 (pg)
25 31.53
50 55.16
100 98.66
200 191.55
Progesterone (ng)
2 2.46
6 6.78
8 8.36
a . . .
Stx determinations (adjusted for procedural losses).
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 differ-
ences 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 deter-
mined for induced and postpartum lactations.
RESULTS A N D DISCUSSION
Validation of Assay Procedures,
Recovery, and Blank Estimates
Displacement curves prepared by assaying
increasing amounts of standard cold estradiol-
1713 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
Recovery
--SE --
.67 126.12
4.48 110.32
7.81 98.66
14.04 95.77
.11 123.00
.43 113.00
.41 104.50
Journal of Dairy Science Vol. 62, No. 7, 1979
1072
o.----~ POST PARTII'-I
o 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 proges-
terone.
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 lacta-
tion 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
NARENDRAN ET AL.
milk paralleled each other after day 6 of
lactation. Total free estrogen in milk probably
was underestimated because estradiol-17t3 stan-
dard 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 parallel-
ism (P<.05), indicating that the differences
between each pair of adjacent estrogen measures
were the same for both groups. But the estrogen
profiles for the two groups were not the same
(P<.001) and the pooled profile for the two
groups was not flat (P<.01). These implied that
estrogen in the two groups, although different,
showed the same pattern of changes in the
period studied. Percentage fat in milk was
correlated significantly with estrogen in milk
during induced (r = .28, P<.001 ) and postpartum
(r =. 19, P<.006) lactations.
Erb et al. (5) reported estrogen at 297 + 95,
310 -+ 111, 144 + 53, 162 + 60, 180 + 73 pg/ml
during days 1, 2, 4, 7, 14, and 42 (commencing
when fluid was first palpated in the gland) of
induced lactation. Total estrogen 714 -+ 292
pg/ml and 334 + 34 pg/ml for days 0 to 2 and 3
to 17 days of induced lactation have been
reported (6). The concentrations for days 0 to
2 of induced lactation are higher than those for
the corresponding period in this study. Monk et
al. (17) and Erb et al. (6) reported high estro-
gens in milk in the immediate prepartum period
(2,142 pg/ml on day 4 prepartum) and days 0
to 2 of normal lactation (1867 -+ 438 pg/ml).
Kesler et al. (15) reported 1630, 350, 334, and
290 pg/ml of milk during 0 (day of calving), 1,
2, and 5 days postpartum in beef cattle. High
estradiol and estrone in serum amounting to
180 and 726 pg/ml at parturition have been
reported by Smith et al. (28), and these declined
to 52 and 115 pg/ml on days 3 and 9 post-
partum. This is in conformity with the trend of
estrogen in postpartum milk. Relationships
have not been reported between estrogen and
fat percentage in milk.
Evidence has been presented for the possible
conversion of estradiol to estrone (5) and
estrone and estradiol-17t3 to biologically inactive
estradiol-17c~ (6) by the cow mammary gland.
Challis and Linzell (4) reported conversion of
estrone to estradiol-1713 in udders of goats, but
 ESTROGEN AND PROGESTERONE IN MILK
l I I ~ I 1t I I7
DAYS CIz LACTATIO~
Figure 2. Progresterone in milk during the first 21
days of induced and postpartum lactations (n=18 per
group).
evidence for a similar conversion has not been
reported for the cow. It is likely that the total
free estrogen in milk was underestimated in this
and other studies (5, 6, 15, 17) in view of
the estrogen conversions in the mammary gland
and the use of estradiol-17/3 standards to
quantify the total free estrogens. Mean estrogen
concentrations in different studies cannot be
compared with accuracy due to variability
between the studies arising from differences in
antibodies, methods of processing milk, and use
of different procedures to measure and adjust
for nonspecific binding.
Progesterone in Induced and
Postpartum Milk
Progesterone in induced milk was higher than
in postpartum milk for the first 21 days of lacta-
tion (Figure 2). In induced lactation, progester-
one 11 + 3 ng/ml on day 1 rose to 22 -+ 9 ng/ml
on day 3 and thereafter fluctuated in the range
of 9 to 16 ng/ml over the rest of the period.
There was a trend for the progesterone concen-
tration in milk to decline as lactation progressed.
In postpartum lactation the progesterone con-
centrations of milk gradually increased from a
low of 4.35 + 1.2 ng/ml on day 1 to 11 + 2
1073
ng/ml on day 21. Progesterone in induced and
postpartum lactations approached each other
towards the latter part of the 3rd wk of lactation.
Progesterone in induced and postpartum
lactation were not parallel, i.e., not mutually
identical (P<.02) as compared by multiple
profile analysis. This implies that progesterone
in milk during induced and postpartum lacta-
tions was significantly different. Percentage fat
was significantly correlated (r = .12, P<.05)
with progesterone only during postpartum
lactation. The progesterone-to-fat ratios in
induced and postpartum lactations were 4.15
and 2.~4.
Erb et al. (5) reported progesterone concen-
trations of 17.6 -+ 2.5, 17.4 + 2.6, 18.7 + 2.6,
I 11.0 -+ 4.2, 5.1 +- 2.2, and 3.9 -+ 1.5 during days
1, 2, 4, 7, 14, and 42 (samples beginning when
fluid first was palpated in the gland) of induced
lactations. These conform with the estimates of
milk progesterone in our study, though the
commencement of the lactation periods is
measured in the two studies by different
standards. Kesler et al. (15) reported progester-
one 1.2 -+ .3, 1.0 -+ .2, 8 -+ .2, and .9 + .2 ng/ml
on days 0 (day of calving), 1, 2, and 5 of
postpartum lactation in beef cows. Erb et al.
(6) reported progesterone 1.4 + .3 ng/ml and .7
+ .1 ng/ml for days 0 to 2 and 3 to 17 of
induced lactations and attributed these small
amounts to low milk yield in the induced cows.
These are much lower than those of our study.
The general trend in the milk progesterone
during postpartum lactation is progressively to
increase with advancing lactation reaching 11 +
2 ng/ml by day 21, whereas Erb et al. (6) and
Kesler et al. (15) reported a decline in milk
progesterone with advancing lactation in the
immediate postpartum period. Smith et al. (28)
reported low progestin concentrations of serum
averaging .6 ng/ml from 0 to 9 days postpartum
in dairy cows. Robertson (22) reported wide
variations in patterns of plasma progesterone in
the immediate postpartum period, and in his
study progesterone rapidly declined to .5 ng/ml
and remained there until resumption of cyclic
activity. Robertson (22) also reported consider-
able variability in the duration of basal proges-
terone, postpartum anestrus period (20 to 60
days) and in the occurrence of one or more
cycles of luteal activity not accompanied by
estrus. Progresterone of milk does not conform
with the blood profile of progesterone during
Journal of Dairy Science Vol. 62, No. 7, 1979
1074 NARENDRAN ET AL.
t h e p o s t p a r t u m period, a n d t h i s m a y b e indica-
tive o f a n active role b y t h e b o v i n e m a m m a r y
g l a n d in u p t a k e a n d m e t a b o l i s m o f p r o g e s t e r o n e
d u r i n g t h e initial stages o f l a c t a t i o n as in the
g o a t (10, 24), c o u p l e d w i t h increasing luteal
activity o f t h e ovary (21). Heap et al. (12)
discussed t h e possibility t h a t t h e CPB assay a n d
r a d i o i m m u n o a s s a y s using less specific a n t i s e r a
m a y b e m e a s u r i n g a y e t u n i d e n t i f i e d com-
p o u n d ( s ) in milk w i t h a n average c o n c e n t r a t i o n
o f 1 t o 2 n g / m l . Such a possibility c a n n o t be
p r e c l u d e d . The d i f f e r e n c e s in t i m e s o f m i l k i n g
(8), m e t h o d o f sample c o l l e c t i o n (12), storage
a n d h a n d l i n g p r i o r t o analysis (19), have
a f f e c t e d p r o g e s t e r o n e in milk a n d c o r r e l a t i o n
b e t w e e n milk f a t a n d p r o g e s t e r o n e . T h e lack of
c o r r e l a t i o n b e t w e e n p e r c e n t a g e fat a n d proges-
t e r o n e in p o s t p a r t u m milk m a y i n d i c a t e involve-
m e n t o f w h e y p r o t e i n o r o t h e r factors as
suggested b y Keller et al. (14). T h e d i f f e r e n c e s
b e t w e e n o u r a n d o t h e r studies f o r m i l k proges-
t e r o n e a n d c o r r e l a t i o n s b e t w e e n milk fat a n d
p r o g e s t e r o n e m a y b e ascribed p a r t l y to t h e s e
factors.
CONCLUSIONS
Extensive m a m m a r y u p t a k e a n d m e t a b o l i s m
(7, 11, 17, 20, 23, 2 4 ) a n d t h e m a m m a r y g l a n d
b e i n g a r o u t e o f steroid h o r m o n e e x c r e t i o n (17)
m a y a c c o u n t for t h e high e s t r o g e n s and proges-
t e r o n e in milk. E s t r o g e n a n d p r o g e s t e r o n e in
i n d u c e d milk, a l t h o u g h h i g h e r t h a n t h o s e in
p o s t p a r t u m milk d u r i n g m o s t o f t h e l a c t a t i o n
periods were c o m p a r a b l e t o t h o s e d u r i n g
c e r t a i n days in p o s t p a r t u m l a c t a t i o n a n d t h o s e
r e p o r t e d in milk b y - p r o d u c t s (8). It seems
r e a s o n a b l e t h a t i n d u c e d l a c t a t i o n milk is as
" s a f e " for h u m a n c o n s u m p t i o n as n o r m a l milk
a n d o t h e r milk b y - p r o d u c t s . T h e c o n c e p t o f
" s a f e t y " as r e l a t e d t o n a t u r a l a n i m a l f o o d s is
largely u n d e f i n e d (16). A r e a s o n a b l e decision
a b o u t the safety o f such a p r o d u c t as milk f r o m
i n d u c e d l a c t a t i o n s can b e r e a c h e d o n l y in t e r m s
o f c o m p a r i s o n w i t h similar a n d t r a d i t i o n a l l y
a c c e p t e d h u m a n foods.
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