DOMESTIC ANIMAL ENDOCRINOLOGY Vol.

7(2):181-190, 1990

CHANGES IN HORMONAL PROFILES DURING THE ESTROUS

CYCLE IN OLD LACTATING BEEF COWS 1'=
R.W. Bryner, M. Garcia-Winder a,
P.E. Lewis, E.K. Inskeep and R.L. Butcher.4
Division of Animal and Veterinary Sciences
and
"Department of Obstetrics and Gynecology,
West Virginia University,
Morgantown, WV 26506

Received June 28, 1989

ABSTRACT
Patterns of concentrations of luteinizing hormone (LH), follicle stimulating hormone
(FSH), progesterone (P~) and estradiol- 17~ (E2) during an estrous cycle were compared
between 15 lactating beef cows 5 to 7 years of age (young) and 15 cows ~_ 12 years
of age (old). Length of estrous cycle did not differ between young and old cows (P =
.06). No differences due to age were found for LH. Patterns of concentrations of P,
during the first 15 days of the cycle, of FSH during days 6 through 12 and of E, during
the follicular phase differed with age (P < .05). An earlier (P < .025) midcycle elevation
of FSH was associated with an earlier rise and greater concentration of E2 (P < .05)
during the follicular phase in old than in young cows. Differences in FSH and P4,
although subtle, were consistent with an earlier or more advanced follicular development
in old cows, leading to greater secretion of EL from the preovulatory follicle.
INTRODUCTION
Fertility declines d u r i n g the latter part o f r e p r o d u c t i v e life and is associated
w i t h alterations in the s e c r e t i o n of g o n a d o t r o p i n s and ovarian h o r m o n e s (1).
With advancing maternal age, there are decreases in fertilization rate, implan-
tation rate and regularity of estrous cycles, as w e l l as increases in e m b r y o n i c
deaths, abortions and d e v e l o p m e n t a l anomalies. In many species, advanced
maternal age leads to r e d u c t i o n s in n u m b e r s o f b o t h primordial and growing
follicles (2, 3, 4). It has b e e n h y p o t h e s i z e d that f e w e r growing follicles w o u l d
lead to a decrease in the p r o d u c t i o n of inhibin, resulting in an increased
secretion o f follicle stimulating h o r m o n e (FSH) (1). An increase in concen-
trations of FSH w o u l d lead to an earlier r e c r u i t m e n t o f follicles and an earlier
p r o d u c t i o n o f estradiol-17~ (E2) b y follicles. Secretion o f FSH has b e e n shown
to be increased in o l d e r rats (5, 6). Elevated levels of FSH and E2 w i t h o u t a
change in luteinizing h o r m o n e (LH) w e r e f o u n d during the early follicular
phase in s e r u m o f w o m e n in the late p r e m e n o p a u s a l p e r i o d c o m p a r e d to
w o m e n 20 to 29 years o f age ('7).
An early and p r o l o n g e d rise in estrogen w i t h i n the follicle (8) c o u l d have
detrimental effects on the o o c y t e itself, decreasing its ability to be fertilized
and d e v e l o p n o r m a l l y (9, 10). Lerner et al. (11) r e p o r t e d a decrease in the
n u m b e r and quality of embryos w i t h increasing age o f Holstein cows. Erickson
(4) and Katska and Smorag (12) r e p o r t e d a significant decrease in the n u m b e r
of follicles in cows w i t h increasing age. It is not k n o w n if the c o n t i n u e d
r e d u c t i o n in the n u m b e r o f follicles w i t h increasing age in the c o w is associated
w i t h alterations in the secretion o f ovarian and pituitary hormones.
Col)yright © 1990 by DOMENDO,INC. 181 0739-7240/90/$3.00
182 BRYNER ET AL.

The purpose of the present study was to determine if profiles of concentrations

of progesterone (P4), E2, FSH and LH differed during estrous cycles of young
and old b e e f cows, and if there were differences in lengths of estrous cycles
between these groups.

MATERIALS A N D METHODS

Thirty lactating b e e f cows of two age groups, 15 cows (9 Hereford, 1 Angus

and 5 crossbred o f predominantly Hereford and Angus breeding) r 12 years
(mean 13.2 _.+ 0.4, hereafter referred to as old) and 15 cows (9 Angus, 2
Hereford and 4 crossbred) 5 to 7 years (mean 6.4 _+ 0.3, hereafter referred
to as young) were obtained from a single commercial farm (while the ratios
of breeds varied, analyses of data showed no breed effects). Cows were main-
rained as a single group, in rotation between two pastures of approximately
20 acres each, containing mixed grasses and limited amounts of white clover.
Beginning May 22, w he n calves averaged 2.9 -+ 0.2 months of age, cows were
observed for estrus (day O) twice daily. Beginning the morning of the second
observed estrus (4.2 _ 0.2 months postpartum) blood samples w ere taken via
jugular v e n i p u n c t u r e dally at 0800 hr. Additional blood samples were taken
at 1400 and 2000 hr on days 9 and 10 to examine more closely the pattern
of FSH, wh ich in a preliminary study appeared to increase earlier in old than
young cows during these days (S.P. Lerner and R.L. Butcher, unpublished).
Starting on day 14 of the estrous cycle, blood samples were taken at 0800 and
2000 hr and assayed daily for P4 to determine the time of regression of the
corpus luteum, defined as the fall in concentration of serum P4 to below 1
ng/ml. From approximately 36 hr after P4 fell bel ow 1 ng/ml until 24 hr after
the detection of estrus, observations for estrus were increased to four times
daily; and blood collections also were increased to four times daily (0200,
0800, 1400 and 2000 hr) to characterize the patterns of LH, FSH and E2 during
the follicular phase. All bl ood samples were placed immediately on ice and
centrifuged within 4 hr of collection; serum was stored at -20 C until assayed.
Estrus was confirmed by a surge of LH and a precipitous decline in concentration
of E2.
H o r m o n a l as s a ys . Concentrations of LH (200 ~tl samples) and FSH (150
~tl samples) in serum were determined by validated radioimmunoassays (13,
14). The intra-and interassay coefficients of variation, respectively, were 7.3
and 6.1% for FSH and 3.8 and 14.3% for LH. Sensitivities of the assays were
.05 rig/tube (n-----5) for LH and .025 ng/tube ( n = 5 ) for FSH. Standards were
NIH-LH-B9 (.70xNIH-LH-S1) and NIAMDD-oFSH-RP-1 (28xUSDA-bFSH-B1).
For the radioimmunoassay of E2 (15), 1.5 ml of serum was extracted twice
with 3 ml of diethyl ether. Estradiol-17~ was separated from other steroids by
column chromatography using Sephadex LH-20 columns, according to pub-
lished procedures (15). Sensitivity of this assay was 1 pg/tube. The intra- and
interassay coefficients of variation each were 5.6%. Concentrations were cor-
rected for recovery (88.5 _-+ 0.4%, n = 1 5 ) .
Concentrations of P4 were determined from 100 ~tl samples that were ex-
tracted twice with 2 ml of p e t r o l e u m ether and assayed according to the
p r o c e d u r e of Sheffel et al. (16). The sensitivity of the assay was 20 pg/tube.
The intra- and interassay coefficients of variation were 6.6 and 7.0% respectively.
Concentrations were corrected for recovery (76.2 _+ 1.2%, n = 1 5 ) .
HORMONAL PROFILES IN OLD COWS 183

Statistical a n a l y s e s . Hormonal profiles were examined by analysis of var-

iance for a split plot design, with age in the main plot and time of sampling
and interactions in the subplot (17). The hormonal profiles examined were
as follows: 1) all four hormones (E2, P~, LH, FSH) during the luteal phase
(from day 1 of the estrous cycle to the sample with a concentration of P4 <
1 ng/ml); 2) all hormones during the follicular phase (end of the luteal phase
to the maximal concentration of LH); 3) P4 from days 1 to 15 (prior to CL
regression); 4) P~ during the 3 days prior to and including the first sample <
1 ng/ml (pattern of luteal regression); 5) E2 from days 2 to 8 (early E2 elevation);
6) E2 from 12 hr before to 24 hr after P4 declined by > 50% (for differences
during the time of follicular selection); 7) E2 from 36 to 12 hr before maximal
concentration of LH (for differences in preovulatory pattern); 8) FSH from
days 6 to 12; 9) LH and FSH from 24 hr before to 24 hr after the maximal
observed concentration of LH. The effects of age on length of estrous cycles,
intervals from estrus to the early rise in E2 and to the rise in FSH at days 7 to
9, and the means and durations of these elevations in Ez and FSH were evaluated
by t-test.
RESULTS
Length of estrous cycle did not differ (P = .06) in old (20.3 - .4 days)
versus young cows (21.3 - .3 days). There was no significant difference in
length of either the follicular or luteal phase between old and young cows.
Neither the patterns nor mean concentrations of LH differed between young
and old cows at any time period during the estrous cycle.
Hormonal profiles in old and young cows throughout the estrous cycle are
shown in Figure 1. In order to account for variations in length of cycle, profiles
are divided into three parts: estrus through day 16 of the cycle, the period of
luteal regression and around the surge of LH.
There was an interaction (P < .05) of age and day of cycle for P~ during
the luteal phase that ranged in length from 17.5 to 21.0 days; so P4 was
reexamined during days 1 through 15 which included the rise and plateau in
P~. Again, there was a significant interaction of age and day for P~ (P < .05)
for this 15-day time period, due to a higher concentration of P~ in young than
old cows during days 14 and 15 of the estrous cycle (Figure 1). The pattern
of decline in P4 during luteal regression did not differ between young and old
COWS.
During the elevation in the serum concentration of FSH from days 6 to 12,
there was a significant interaction of age and day of cycle (P < .05) (Figure
2). Since before day 7, FSH was less than .7 ng/ml in all cows, this value was
used to assess time of increase in FSH. The time period from estrus to the first
sample with FSH > .7 ng/ml was shorter (P < .025) in old (8.9 - .2 days)
than in young cows (9.7 +-- .2 days). Neither the duration of this rise in FSH
nor the mean concentration of FSH during days 6 to 12 differed due to age.
The concentration of FSH rose again during the latter part of the luteal phase
in 18 of 30 cows. There was no difference due to age in the proportion of
cows that showed this rise of FSH in the late luteal phase (8 old and 10 young
COWS).
During the period from 24 hr before to 24 hr after the preovulatory surge
of LH, concentration of FSH did not differ due to age. Based upon these data
and those on day 1 of the cycle, both old and young cows had a second peak
of FSH approximately 24 hr after the maximal concentration of the Hi surge.
184 B R Y N E R E T AL.

70
50
----"10

A
4.8

.4

~'~61 j
~4
~2

0 2 4 6 8 10 !2 14 16 -! 0 1 -1 0 1
Days from Daysfrom
Day of estrouscycPe decline of LH peak
>50% in P4 concentration

Fig. 1. Profiles of LH, FSH, progesterone and estradiol-1713 in old ( ) and young (........ )
lactating beef cows during the estrous cycle (n----15). Each profile contains 37 data points, and
t h e shaded areas are -.+ SEM. To account for variations in length of estrous cycles, the patterns
are divided into days 0 to 16, 1.5 days before to 1.5 days after P4 declined > 50% and 1.5 days
before to 1 day after the peak LH concentrations. Note expanded time scale around times of P~
decline and the LH surge.

Patterns o f E2 during days 1 t h r o u g h 15 of the estrous cycle w e r e similar

for old and y o u n g cows. C o n c e n t r a t i o n s o f E2 b e g a n to rise on days 3 or 4 o f
the estrous c y c l e (Figure 3), r e a c h e d a m a x i m u m o f 3.1 p g / m l for y o u n g cows
and 2.9 p g / m l for old cows on the day 5 o f the c y c l e and then d e c l i n e d to
basal levels b y day 7. The timing, duration or m e a n c o n c e n t r a t i o n of this
elevation in E2 d u r i n g the early part o f the luteal phase did not differ b e t w e e n
y o u n g and old cows. Four old and three y o u n g c o w s e x h i b i t e d an increase in
c o n c e n t r a t i o n o f E2 b e t w e e n days 13 and 18; and the elevation lasted o n l y 12
to 48 hr.
The m e a n c o n c e n t r a t i o n o f E2 d u r i n g regression o f the c o r p u s l u t e u m (12
hr p r i o r t h r o u g h 24 hr after the first b l o o d sample in w h i c h P4 was d e c r e a s e d
to < 2 n g / m l ) was greater in old than y o u n g c o w s (P < .05; Figure 4).
C o n c e n t r a t i o n s of E2 also w e r e elevated in old c o m p a r e d to y o u n g c o w s (P <
.05) d u r i n g the 36 hr p r i o r to the maximal c o n c e n t r a t i o n of LH d u r i n g the
o v u l a t o r y surge (Figure 4).
DISCUSSION
The general patterns o f h o r m o n a l secretions w e r e similar in old and y o u n g
cows, but there w e r e significant alterations in the profiles o f E2 and FSH d u e
HORMONAL PROFILES IN OLD COWS 185

1.0 * Old
.... ° .... Y o u n g ~ _
O.9

I I I I I I i

6 7 8 9 10 11 12
Days of estrous cycle
Fig. 2. Patterns of concentrations of FSH in young and old cows during the elevation at days 6 to
12 ( n = 15), The rise occurred earlier in old than young cows (P < .025). Mean square for error
for testing age by day interaction was 0.035.

to age. Similar changes in profiles in old rats (6, 8, 18) have been associated
with decreased numbers of developing follicles (19) and increased size and
E2 content of these follicles (8, 20). Also, the secretion of inhibin is reduced
(21), resulting in early or increased secretion of FSH, which results in early
development of follicles during the estrous cycle (20). Increased secretion of
FSH and E, during the follicular phase of the menstrual cycle has been reported
in women during late reproductive life (7).

3.5 --
- Old
..... • .... Y o u n g
3.0 --

E
Q.
2.5 -
m
_o
"0
m

ul 2.0 --

1.5 --

I I I I I ]

2 3 4 5 6 7 8
Days of estrous cycle
Fig. 3. Patterns of concentrations of E= in young and old cows early in the estrous cycle ( n = 15).
Age by time interaction (patterns) were not different (P > .05).
186 BRYNER ET AL.

I " Old
7-1
i - --" .... Young
I
E 6
O}

.o 5
.m
4
I.IJ ,/ T ."

/ ..

/ /
! I / /
-12 0 12 24 -36 -24 -12

Hours from P4 decline Hours before LH surge

Fig. 4. Patterns of concentrations of E2 in young and old cows during the time of luteal regression
(0 hr ~= P, < 2 ng/ml) and prior to the maximal concentration of the LH surge (n = 15).
Concentrations of E2 were greater in old than young cows during both periods (P < .05). Mean
squares for error were 8.52 for the period of luteal regression and 4.72 for 12 to 36 hr before
the LH surge.

E s t r a d i o l . In the p r e s e n t study, the elevation o f E2 in p e r i p h e r a l b l o o d early

in the estrous cycle (Figure 3) fits well w i t h the r e p o r t e d rise in E2 in the
utero-ovarian vein of cows at 3 to 7 days after the LH surge (22). This rise in
E2 is from the first d o m i n a n t follicle of the estrous c y c l e (23, 24, 25, 26).
The time o f this rise and fall in c o n c e n t r a t i o n o f E2 was not significantly different
in y o u n g and old cows.
The h i g h e r c o n c e n t r a t i o n o f E2 in old than y o u n g cows d u r i n g luteal regression
c o u l d r e p r e s e n t an earlier g r o w t h of the p r e o v u l a t o r y follicle (Figure 4).
Likewise, the h i g h e r c o n c e n t r a t i o n o f E2 during the p e r i o d o f 12 to 36 hr p r i o r
to the maximal c o n c e n t r a t i o n o f the LH surge c o u l d r e p r e s e n t a more advanced
stage o f f o l l i c u l a r d e v e l o p m e n t (Figure 4). The maximal c o n c e n t r a t i o n o f E2
did not differ d u e to age. While c o n c e n t r a t i o n s o f LH did not differ at any
t i m e b e t w e e n y o u n g and old cows, concentrations of E2 w e r e elevated early
in relation to the time o f the LH surge in old cows. T h e r e may be a decreased
sensitivity to positive f e e d b a c k o f E2 in old cows, as has b e e n r e p o r t e d for
middle-aged rats ( 2 7 ) . Butcher and Page (1) r e p o r t e d an earlier rise and
p r o l o n g e d elevation o f the p r e o v u l a t o r y rise o f E 2 in old c o m p a r e d to y o u n g
rats. In middle-aged rats that still maintained 4-day estrous cycles, there w e r e
larger follicles c o n t a i n i n g m o r e Ez early in the cycle than in y o u n g rats at
similar times ( 2 0 ) .
E x p e c t e d g r o w t h of a s e c o n d d o m i n a n t follicle after m i d c y c l e (25, 26) was
not reflected by an increase in c o n c e n t r a t i o n o f E2 in p e r i p h e r a l circulation
o f most cows in the p r e s e n t study• Failure to d e t e c t an increase in circulating
E2 at this time is not surprising, considering that the increase in c o n c e n t r a t i o n
o f Ez in the utero-ovarian serum was m u c h less during g r o w t h o f the s e c o n d
( 2 8 ) than o f the first and third d o m i n a n t follicles ( 2 2 ) . This b r i e f rise in E2
in o n l y 9 of 30 cows was not associated with age or w i t h cows that had a rise
HORMONAL PROFILES IN OLD COWS 187

in FSH late in the luteal phase, and the E2 rose later than expected based on
the reported time at which growth of the second dominant follicle begins (25,
26). The low rate of secretion of E2 during growth of the expected second
dominant follicle, as compared to marked elevation of E2 during the follicular
phase, likely is due to suppression of E2 by P4 (13). The first dominant follicle
is reported to persist at least to day 14 to 16 of the estrous cycle (25, 26),
but termination of secretion of E2 by day 7 probably results from its suppression
by increasing concentrations of P4.
FSH. The rise in FSH about 24 hr after the LH surge is in agreement with
reports in cows (29), sheep (30) and rats (9), and likely reflects a decline in
inhibin as a result of the preovulatory LH-FSH surge. Elevated FSH at the time
of the LH surge and again 24 hr later, is likely involved in recruitment of the
first dominant follicle and the rise in E2 secretion early in the estrous cycle.
The rise in concentrations of FSH during days 7 to 9 of the estrous cycle
(Figure 2) was closely associated with the decline in E2 during days 5 to 7
(Figure 3). Secretion of inhibin (not measured in this study) by follicles is
known to be associated with the secretion of E2 and would be expected to
decline with the decline in E2. The decline of follicular function during days
5 to 7 is proposed as a cause of the rise in serum FSH during days 7 to 9.
The second dominant follicle, which is first recognized with ultrasound imaging
around days 12 to 13 of the estrous cycle (25, 26), likely is selected by the
elevation of FSH during days 6 to 12. The earlier elevation in FSH in old cows
w o u l d be expected to produce an earlier selection of the second dominant
follicle. We propose that an earlier selection of the second dominant follicle
in old cows is in turn responsible for an earlier and more advanced development
of the ovulatory follicle, which results in a greater elevation in the concentration
of E2 in old than in young cows as P4 declines with regression of the corpus
luteum. In some cows the second dominant follicle is the ovulatory follicle,
while in others a third dominant follicle ovulates (25, 26). The rise in
concentration of FSH in 18 of 30 cows late in the luteal phase likely was due
to atresia of the second dominant follicle. This late luteal phase increase of
FSH would select the third dominant follicle, which could be at a more advanced
stage of development in old cows due to recruitment by the earlier rise in
FSH at days 6 to 12 of the cycle. Future studies utilizing ultrasound observations
in conjunction with concentrations of inhibin, FSH and E2 are needed to test
these proposed interactions in the recruitment and selection of the dominant
follicles.
C o n c l u s i o n s . The altered profiles of E2 and FSH in old cows probably are
responses to decreased numbers of growing follicles and changes in the hy-
pothalamus (1). These changes likely contribute to decreasing reproductive
efficiency through alterations of both the oocyte and intrauterine environment
by elevations in Ez (10). There was a decrease in fertilization rate and a decrease
in quality of the embryos from old cows that were superovulated (11).
ACKNOWLEDGEMENTS AND FOOTNOTES

~Published with the approval of the director of the West Virginia Agricultural and Forestry
Experiment Station as Scientific Paper No. 2179.
aSupported by a grant from Select Sires, Inc. and by Hatch Project 224. The authors thank Dr.
G.D. Niswender, Colorado State Univ., for antiserum to LH; Dr. L.E. Reichart, Jr., Albany Medical
College, for purified LI-I; NIDDKD and the National Hormone and Pituitary Program for immune-
reagents for FSH assays; Drs. W.V. Thayne and E.C. Townsend for assistance with statistical analyses;
Diana Kirkpatrick-Keller for assays of steroids; Dr. Steven Lerner for computer graphics.
188 BRYNER ET AL.

3Present address: Centro de Ganaderia, Colegio de Postgraduados, Chapingo, Mexico 56230.

4Correspondence and reprint requests to: Dr. R.L. Butcher, Department of Obstetrics and Gyne-
cology, Health Sciences Center North, West Virginia University, Morgantown, WV 26506-6302.

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