Animal Reproduction Science, 21 (1989) 1-10 1

Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Ovarian Control of Gonadotrophin Secretion in the

Prepubertal Heifer

S.E. DODSON, B.J. MCLEOD*, W. HARESIGN, A.R. PETERS**, G.E. LAMMING and
D. DAS***
A.F.R.C. Research Group on Hormones and Farm Animal Reproduction, University of
Nottingham School o/Agriculture, Sutton Bonington, Loughborough, Leics. LE12 5RD
(Great Britain)
{Accepted 5 June 1989)

ABSTRACT

Dodson, S.E., McLeod, B.J., Haresign, W., Peters, A.R., Lamming, G.E. and Das, D., 1989. Ovar-
ian control of gonadotrophin secretion in the prepubertal heifer. Anita. Reprod. Sci., 21: 1-10.

In order to investigate the development of ovarian feedback control of gonadotrophin secretion

in the immature animal, three groups of eight Hereford × Friesian heifers were bilaterally ovariec-
tomized at either 2, 6 or 12 weeks of age. Blood samples were collected for gonadotrophin analysis
( 15-min intervals for 6 h) on days - 1, 4, 7, 14, 28, 55 and 110 relative to ovariectomy. The ovaries
from these animals showed that follicular development was established soon after birth and the
follicles were producing oestradiol. In all animals, ovariectomy was followed by a rise in plasma
gonadotrophin concentrations although the response was delayed in heifers ovariectomized at 2
weeks compared to those ovariectomized at 6 or 12 weeks of age. Mean LH concentrations were
significantly elevated 14, 7 or 4 days after ovariectomy and mean FSH concentrations were sig-
nificantly higher than pre-treatment values 28, 7, or 4 days post-ovariectomy in heifers ovariec-
tomized at 2, 6 or 12 weeks of age, respectively. The results suggest that an inhibitory mechanism,
of ovarian origin, that controls gonadotrophin secretion develops between 2 and 12 weeks of age
in the heifer.

INTRODUCTION

Gonadotrophin secretion in the prepubertal heifer is highly sensitive to the

negative feedback effects of ovarian steroids. For example, a rapid increase in

*Present address: A.F.R.C. Institute for Grassland and Animal Productions, Animal Grassland
Research Station, Hurley, Maidenhead, Berks, SL6 5LR (Great Britain)
**Hoechst UK Ltd., Walton Manor, Milton Keynes MK7 7AJ (Great Britain)
***Department of Animal Genetics and Breeding, College of Veterinary Science, Assam Agricul-
tural University, Khanapara, Gauhati-781 022, Assam {India)

0378-4320/89/$03.50 © 1989 Elsevier Science Publishers B.V.

2 S.E. DODSON ET AL.

gonadotrophin secretion occurs after ovariectomy, which is prevented by low

doses of oestradiol (Day et al., 1984; Moseley et al., 1984). In a recent study
investigating the changes in gonadotrophin secretion from birth to puberty,
mean plasma LH concentrations decreased between birth and 15 weeks of age,
due largely to a fall in basal LH concentrations (Dodson et al., 1988). There-
after, mean LH concentrations increased. The decrease in mean LH concen-
trations observed between birth and 15 weeks of age could reflect the devel-
opment of an ovarian inhibitory control over the hypothalamic-pituitary unit.
Alternatively, gonadotrophin secretion may change independently of ovarian
steroids at this time. The present experiment was designed to determine
whether there is an age-related change in the timing of rises in gonadotrophin
secretion following ovariectomy, and thus the removal of ovarian steroids, dur-
ing the first 12 weeks of life.

MATERIALSAND METHODS

Animals
24 autumn-born Hereford × Friesian heifers were bought as 3-day-old calves
and reared on milk substitute and concentrates. After weaning at 12 weeks the
calves were kept as one group and fed concentrates and hay. They were housed
under conditions of natural daylength and temperature throughout the trial.

Treatment
The heifers were randomly allocated to one of three groups of eight animals,
to be bilaterally ovariectomized at either 2 weeks of age (Group 1), 6 weeks
(Group 2 ) or 12 weeks (Group 3). One day before ovariectomy the heifers were
removed from the main group and penned individually. They were returned to
the main group, 8 days post-ovariectomy. Ovariectomy was performed under
general anaesthesia, induced by 5% sodium thiopentone i.v. (Intraval sodium;
May & Baker Ltd., Essex), and maintained with halothane (Fluothane, ICI
plc, Cheshire) and nitrous oxide in oxygen. The ovaries were removed through
a ventral mid-line incision.
After removal, the ovaries were weighed, the diameters of all visible follicles
measured, and the fluid from each follicle was aspirated and stored in buffer
at - 20 ° C until analysed for oestradiol concentrations.

Collection of blood samples

After insertion of a jugular catheter, blood samples (2ml) were collected at
15-rain intervals for 6-h periods on days - 1, 4, 7, 14, 28, 55 and 110, relative
to ovariectomy. Plasma was stored at - 20 ° C until assayed.
CONTROL OF GONADOTROPHIN SECRETION IN THE HEIFER 3

Hormone radioirnmunoassays
Plasma LH concentrations were measured by the method of Webb et al.
(1977). The limit of sensitivity was 0.33 n g / m l and the inter- and intra-assay
coefficients of variation were 7.7% and 8.4%, respectively.
Plasma samples were assayed for FSH by the method of Webb et al. (1980).
The limit of sensitivity was 11 ng/ml. Inter- and intra-assay coefficients of
variation were 13% and 11%, respectively.
Follicular fluid oestradiol was measured using the procedure of Foxcroft et
al. (1984). The mean extraction efficiency was 86 + 6.2% and the limit of sen-
sitivity of the assay was 50 pg/ml. Inter- and intra-assay coefficients of vari-
ation were both < 15%.

Analysis of data
An LH episode was defined by the following criteria: (a) an increase of >/4
times the coefficient of variation of duplicate pairs above the preceding base-
line value, (b) at least 2 points between the peak and the succeeding trough or
baseline, and (c) a rate of decline in LH concentration after the peak consis-
tent with the half-life of the hormone. Basal LH was calculated as the nadir
between LH episodes and LH episode amplitude as the peak concentration of
each episode, regardless of baseline concentration. Plasma FSH concentra-
tions were not analysed for episodes.
All data except LH episode frequency were analysed by analysis of variance,
and differences in the rates of change in hormone concentrations with time
were compared using polynomial regression analysis. LH-episode frequency
was compared by analysis of deviance.

RESULTS

The average daily liveweight gain of the heifers was 0.70 kg/day. The rate of
gain did not vary significantly between treatment groups. Mean body weights
at ovariectomy, which varied significantly (P < 0.001 ) with age, are shown in
Table 1.
Mean ovarian weight was greater ( P < 0.05) at 6 than at 2 weeks of age, but
did not differ between 6 and 12 weeks of age (Table 1). The distribution of
follicle sizes within age groups are shown in Fig. 1. The number of follicles >
2 m m increased with age, and was greater (P < 0.05) in animals ovariectomized
at 6 and 12 weeks of age compared to those ovariectomized at 2 weeks. How-
ever, the total number of follicles per heifer was not different between age
groups.
Mean follicular fluid volume and oestradiol content of the four largest fol-
licles from each animal are shown for each group in Table 1. Both follicle di-
ameter and fluid volume were greater ( P < 0.001 ) in heifers ovariectomized at
6 compared with 2 weeks of age, but these parameters did not differ between
animals ovariectomized at 6 and 12 weeks. Follicular oestradiol concentration
4 S.E. DODSONErrAL.

TABLE 1

Comparison of bodyweight, ovarian weight and follicle characteristics in groups of eight heifers
ovariectomized at either 2 weeks of age, 6 weeks of age or 12 weeks of age

Age Body Total Mean of the four largest follicles

(weeks) weight ovarian
(kg) weight Diameter Fluid vol. Oestradiol
(g) (mm) (gl) conc.
(ng/ml)

2 56 1.92 2.47 13.3 3.74

6 72 4.96 6.03 84.6 4.18
12 96 4.42 6.16 122.8 10.20
s.e.d. 12 2.69 1.33 45.7 6.16

[] group I ( 2 weeks )
[] group 2 (6 meeks)
gn group 3 (12weeks}

~0-
E
c
m
30-
/

/
I o.e.d.
20- /
i
o
J
10-

O-
/
/
/
A
2-t+
j
5-8 >8
Follicle diameter (mm-)
Fig. 1. Distribution of follicles ( < 2 mm, 5-8 mm and > 8 m m diameter) in groups of eight heifers
ovariectomized at either 2, 6 or 12 weeks of age. The s.e.d, indicated is for comparison of follicles
of the same size between age groups.

was positively correlated with follicle size (P < 0.01 ), but was not significantly
different between age groups. In addition, there was no significant correlation
between the mean follicular fluid oestradiol concentration from the four larg-
est follicles, and gonadotrophin concentrations on the day before ovariectomy.
There were no significant differences between age groups in mean LH con-
centrations during the pre-treatment period. However, the rate of increase in
mean LH concentrations following ovariectomy varied significantly ( P < 0.001 )
with age (Fig. 2 and 3). For example, in heifers ovariectomized at 2 weeks of
age, overall mean LH concentrations did not significantly increase until 14
days post-ovariectomy, whereas this occurred by 7 and 4 day post-ovariectomy
in those heifers ovariectomized at 6 or 12 weeks of age, respectively. In addi-
tion, mean LH concentrations increased throughout the 110 days of study in
CONTROL OF GONADOTROPHIN SECRETION IN T H E HEIFER

S•,
= a ) between within
•-~ ~ group group
{ ~ 0 J

® b)

I z

, t I I
e)
"~ 400"

-r

200. D Group

• Group 2
~E
Group 3
-! 4 7 14 28 55 110

Days after ovariectomy

Fig. 2. Parameters of gonadotrophin release at different times relative to ovariectomy in groups

of eight heifers ovariectomized at either 2 weeks of age, 6 weeks of age or 12 weeks of age. {a)
mean LH episode frequency, (b) mean LH episode amplitude, (c) mean basal LH concentrations,
(d) overall mean plasma LH concentration, (e) overall mean plasma FSH concentration. + and
* indicate that the post-ovariectomy mean values are significantly higher than the preovariectomy
value at P < 0.1 and P < 0.05 (at least), respectively. The appropriate s.e.d, for comparing mean
values at different times within a group and at the same time between groups are indicated.

heifers o v a r i e c t o m i z e d a t 2 or 6 weeks. B y w a y o f c o n t r a s t , in a n i m a l s o v a r i e c -
t o m i z e d a t 12 w e e k s m e a n L H c o n c e n t r a t i o n s i n c r e a s e d u p to d a y 55 b u t t h e n
d e c r e a s e d s i g n i f i c a n t l y ( P < 0.01 ) b y d a y 110 p o s t - o v a r i e c t o m y (Fig. 2d).
T h e initial i n c r e a s e in m e a n L H c o n c e n t r a t i o n s following o v a r i e c t o m y re-
s u l t e d f r o m a n i n c r e a s e in L H e p i s o d e f r e q u e n c y (Fig. 2 a ) . H o w e v e r , t h e p a t -
t e r n o f c h a n g e in L H e p i s o d e f r e q u e n c y w i t h t i m e w a s s i g n i f i c a n t l y d i f f e r e n t
6 S.E. DODSONET AL.

800"
a)
~6oo
C
7_400
C9

" 200
o
9

E 6-
-&
C

-r
_J
3-

O-
i 7 r - - 7 [ i f - - 1 i - - 1 i 7
t
F - -

800-

E 600- b)

,,~ 200
°
~.00-

0
L
o~
;--2; id £; ;; ;; ;; ;
Time (hours)
-I t, 7 I L, 28 55 100
Days after ovariecfomy
Fig. 3. Plasma LH and FSH profiles from two individualheifers followingovariectomyat either
(a) 2 weeks of age or ( b ) 12 weeks of age.

between age groups. There was a delayed increase in L H episode frequency in

heifers ovariectomized at 2 weeks of age compared with heifers ovariectomized
at either 6 or 12 weeks of age. Furthermore, the initial increase in L H episode
frequence was delayed in heifers ovariectomized at 6 weeks of age compared
with those ovariectomized at 12 weeks of age (Fig. 2a).
As overall mean L H concentrations increased with time post-ovariectomy,
CONTROL OF GONADOTROPHINSECRETION IN THE HEIFER 7

there was also a significant increase in basal LH concentrations in all treat-

ment groups (Fig. 2c). The timing of the increase in basal LH concentrations
following ovariectomy, varied significantly (P<0.05) between treatment
groups. Although basal LH concentrations did not differ significantly with age
prior to ovariectomy, basal LH concentrations were significantly higher
( P < 0.05) than pre-ovariectomy values by 28, 14 or 7 days post-ovariectomy
in heifers ovariectomized at 2, 6 and 12 weeks respectively. Furthermore, basal
LH concentrations continued to increase throughout the study in heifers
ovariectomized at either 2 or 6 weeks of age, but in animals ovariectomized at
12 weeks, basal LH concentrations were lower ( P < 0.001 ) on day 110 than on
day 55 post-ovariectomy.
Since episodic LH release was recorded in only three heifers during the pre-
treatment period, and all these animals were in the oldest age group (Group
3), between-group differences in episode amplitude have been analysed from
day 4 post-treatment. Changes in the amplitude of LH episodes varied signif-
icantly between groups (P < 0.05 ). LH episode amplitude was not significantly
greater than day 4 values until day 110 or day 28 respectively for heifers ovari-
ectomized at 2 and 6 weeks of age. However, in heifers ovariectomized at 12
weeks of age, LH episode amplitude was never significantly different from that
recorded on day 4 post-ovariectomy (Fig. 2b).
Mean FSH concentrations recorded over the pre-treatment period did not
vary significantly with age, but the response to ovariectomy was again signif-
icantly different (P<0.01) between groups (Fig. 2e). Mean FSH concentra-
tions were significantly higher than pre-treatment values by 28, 7 and 4 days
post-ovariectomy in heifers ovariectomized at 2, 6 and 12 weeks respectively.
Plasma FSH concentrations continued to increase throughout the period of
study in heifers ovariectomized at either 2 or 6 weeks but following an initial
increase up to day 55 had declined significantly (P<0.01) by day 110 in ani-
mals ovariectomized at 12 weeks. Following ovariectomy, changes in mean FSH
and mean LH concentrations were highly correlated ( P < 0.001) in all treat-
ment groups.
DISCUSSION

Ovaries from heifers of the same age showed considerable variation in size
and follicular development as in previous reports (Casida et al., 1935; Erick-
son, 1969; Onuma and Foote, 1969). However, in contrast to other reports
(Erickson, 1966; Desjardins and Hafs, 1969), the total number of ovarian fol-
licles did not change with age. The proportion of large follicles did, however,
increase with age; for example, most 12-week-old heifers possessed at least one
follicle > 8 mm diameter and containing measurable levels of oestradiol, pos-
sibly suggesting that the ovaries were functional but lacking the gonadotrophin
stimulus necessary for the final stages of follicle maturation and ovulation.
This view is supported by the fact that ovulation can be induced in 8-week-old
8 S~.DODSON ETAL.

calves treated with gonadotrophins although such ovulations are not followed
by normal luteal activity (Seidel et al., 1971 ).
Ovariectomy resulted in a transition in the pattern of gonadotrophin secre-
tion from one of infrequent LH episodes and low mean LH and FSH concen-
trations, to one of high frequency LH episodes and elevated mean LH and FSH
concentrations. The rate with which gonadotrophin secretion increased was
dependent on the age of the animal at ovariectomy. The response was delayed
in heifers ovariectomized at either 2 or 6 weeks of age compared to heifers
ovariectomized at 12 weeks of age, consistent with previous findings in the
lamb (Foster et al., 1972), the bull (Bass et al., 1977; Wise et al., 1987 ), the
pony (Wesson and Ginther, 1979) and the heifer (Anderson et al., 1986). Fur-
thermore, the response to ovariectomy was delayed in heifers ovariectomized
at 6 weeks of age compared to 12 weeks of age, perhaps indicating that ovarian
negative feedback control over gonadotrophin secretion develops progressively
rather than abruptly between 2 and 12 weeks of age.
The increase in plasma LH concentrations following ovariectomy was ini-
tially due to an increase in LH episode frequency which was then followed by
an increase in basal LH concentrations. In heifers ovariectomized at either 6
or 12 weeks of age, LH episodes occurred at the rate of 1/h by 55 days post-
ovariectomy, whereas this frequency was not evident until day 110 in animals
ovariectomized at 2 weeks of age. Similar observations have been made in heif-
ers ovariectomized at 8, 28 and 36 weeks of age (Moseley et al., 1984; Day et
al., 1986), although LH concentrations were maximal by day 8 post-ovariec-
tomy in older prepubertal animals (Kiser et al., 1981 ), indicating that a further
change in the ovarian control over gonadotrophin release probably occurs be-
fore puberty.
The present results indicate that the ability to produce high frequency LH
episodes after ovariectomy in the heifer is not present until approximately 6
weeks of age, and may therefore reflect a maturational change within the hy-
pothalamic-pituitary axis. Alternatively, it may be held in check by some other
source since factors withing the central nervous system are known to inhibit
episodic LH release in the neonate (Levasseur, 1979 ). Further studies involv-
ing ovariectomy and steroid replacement are required to distinguish between
these possibilities.
A large degree of between-animal variation was apparent in the gonadotro-
phin response to ovariectomy, particularly in heifers ovariectomized at 2 and
6 weeks. Generally there was a more rapid response following the removal of
ovaries that contained large follicles. For example, most 2-week-old heifers had
no follicles > 2 mm and no increase in plasma LH concentrations until at least
14 days post-ovariectomy. However, in one 2-week-old heifer in which there
were three follicles > 3 ram, gonadotrophin concentrations increased by day 4
post-ovariectomy. Furthermore, the only heifer in Group 2 (6 weeks of age)
which failed to show an increase in LH episode frequency by 4 days post-ovari-
CONTROL OF GONADOTROPHIN SECRETION IN THE HEIFER 9

ectomy, was the only animal possessing no follicles > 4 mm diameter at the
time of ovariectomy. It is therefore possible that steroid inhibition of LH se-
cretion develops coincidentally with the first large ovarian follicles.
In heifers ovariectomized at 12 weeks, overall mean plasma gonadotrophin
concentrations on day 110 post-ovariectomy were suppressed relative to those
on day 55 post-ovariectomy, largely due to a decrease in basal LH concentra-
tions. Similar changes in gonadotrophin secretion have been reported follow-
ing ovariectomy in 36-week-old heifers (Day et al., 1986). The reason for this
decrease in gonadotrophin secretion between 55 and 110 days post-ovariec-
tomy in 12-week-old heifers is not known. However, possible explanations may
include a change in pituitary responsiveness to GnRH following continuous
exposure to high frequency GnRH pulses, which results in submaximal gona-
dotrophin secretion, or a maturational change within the hypothalamic-pitui-
tary axis coincident with the normal time of pubertal onset.
In a parallel study in this laboratory, overall mean LH concentrations, basal
LH concentrations and overall mean FSH concentrations decreased between
3 and 15 weeks of age in the heifer, despite increasing LH episode frequency
(Dodson et al., 1988). These results taken together with the present study
suggest that a maturational change occurs in the hypothalamic-pituitary axis
between 2 and 12 weeks of age whereby the ovary begins to control gonadotro-
phin secretion, and that the decrease in gonadotrophin secretion between 3
and 15 weeks in the entire heifer (Dodson et al., 1988) results from an increase
in negative feedback influences from the ovary.

ACKNOWLEDGEMENTS

We thank Dr. M.G. Hunter for undertaking the measurements on ovarian

follicles, the A.F.R.C. for financial support; and NIAMKDD Bethesda, U.S.A.
for standard LH and FSH preparations.

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