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This is to certify that the
dissertation entitled
Brain and Pineal Gland Mediation of

Photoperiod-Induced Hormone Concentrations in Cattle

presented by

E ward Peter Stanisiewski

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal Science

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BRAIN AND PINEAL GLAND MEDIATION OF PHOTOPERIOD-INDUCED

HORMONE CONCENTRATIONS IN CATTLE

BV

Edward Peter Stanisiewski

A DISSERTATION

Submitted to
Michiaan State Universitv
in partial Fulfillment of the requirement
for the decree 0F

DOCTOR OF PHILOSOPHY

Department of Animal Science

1987

ABSTRACT
BRAIN AND PINEAL GLAND MEDIATION OF PHOTOPERIOD-INDUCED
HORMONE
CONCENTRATIONS IN CATTLE
by

Edward Peter Stanisiewski

Concentrations of prolactin (PRL) in serum were 2 to 3—
fold greater in prepubertal bulls exposed to 24 (24 Liaht:0
Dark) or 16 (16L:8D) h of daily light as compared with
8L:160. However, PRL attained maximum concentrations
approximately 3 wk later in calves exposed to 24L:0D.

In other bulls, PRL was 2.5-fold greater after 6-wk
exposure to 16L:8D compared with 8L:16D, while testosterone
was concurrently increased 2-fold by 16L:80. However, after
6 additional wk of repetitive application of 16L:8D bulls
became refractory in terms of PRL while testosterone
concentrations plateaued. Photoperiod did not affect serum
luteinizing hormone concentrations. Shifting from 8L:16D to
161:80 increased serum concentrations of PRL equally in
castrated and gonadally intact males.

Dopamine concentrations in anterior pituitary glands
were greater in calves exposed to 8L:16D compared with
16L:8D, while plasma catecholamine concentrations were not
affected by photoperiod.

In other experiments, serum melatonin concentrations
varied episodically and were 60 to 405% greater during dark

than light periods, while 24L:0D abolished the nocturnal

increase. Exposure to 8-h of darkness coincidently elevated
serum melatonin, whereas melatonin is elevated for only the
first 12 to 14-h of a 16-h dark period. Serum melatonin is
probably not responsible for photoperiOd-induced changes in
serum PRL since PRL concentrations are equivalent in bulls
infused daily with melatonin for 8 or 16-h.

Pinealectomy (PX) of bull calves does not abolish
photoperiod-induced changes in serum PRL concentrations:
however, testosterone concentrations in PX bulls are lower
at 20 wk of age than in sham-PX bulls.

It is concluded that photoperiod-induced changes in PRL
concentrations does not involve melatonin, the pineal gland
or the testes, but may be mediated by dopamine in
prepubertal bull calves. Serum PRL concentrations in calves
become refractory to prolonged 16L:8D photo-stimulation.
Finally, long-days hasten puberty onset in bulls and the

pineal gland may be involved.

ACK NOWLEDGMENTS

I wish to express appreciation to my maior professor,
Dr. H. A. Tucker, as well as the other members of my
committee, Drs. T. A. Ferris, R. W. Mellenberger, R. A.
Merkel and D. R. Romsos, for their assistance and guidance
in transforming a few ideas into the substance of this
dissertation.

I also wish to thank Drs. N. K. Ames, K. Demarest, J.
L. Gill, P. J. Karsch and Mr. L. T. Chapin for their
invaluable help with methololoqies and logistics. To Miss
B. A. Sheathelm, thank you once again for your time and

effort in typing this dissertation.

TABLE OF CONTENTS

Page
LIST OF TABLES........................;............... iv
LIST OF FIGURES....................................... v
INTRODUCTION........................................... 1
REVIEW OF LITERATURE................................... 3

A. Introduction..................................... 3
B. Seasonal and Photoperiod-Induced
Changes in Hormones.............................. 3
1. Prolactin.................................. 3
2. Gonadotropins.............................. 5
3. Steroid Hormones........................... 7
C. Hypothalamic Control of Prolactin Secretion...... 8
D. Seasonal and Pituitary Gland Control

of Gonadal Function............................. 10
E. Puberty.......................................... l4
1. Definitions................................ 14
2. Endocrine Changes Associated with
Onset of Puberty.......................... 16
3. Neuroendocrine Effects on Puberty.......... 18

4. Manipulation of Puberty Onset.............. 20
F. Light Perception and Endocrine Changes........... 23

1. Functional Anatomy......................... 27
2. Pineal Gland Function...................... 28
3. Melatonin Synthesis and Secretion.......... 31
4. Characteristics of Melatonin............... 32
5. Biological Effects of Melatonin............ 32
G. Objectives....................................... 34

CHAPTER 1. Effect of Continuous Light on
Prolactin Secretion in Prepubertal

Holstein Bulls............................ 36
Introduction................................... 37
Materials and Methods.......................... 38
Results........................................ 40
Discussion..................................... 41

CHAPTER 2. Effect of Photoperiod and Castration
on Prolactin, Testosterone and Luteinizinq
Hormone Concentrations in Male Calves..... 49

ii

Introduction................................... 50
Materials and Methods.......................... 50
Results........................................ 53
Discussion..................................... 60

CHAPTER 3. Prolactin and Cathecholamine Concentrations

in Blood and Pituitaries of Prepubertal

Bulls Exposed to 16 or 8 Hours of Light

Per Day................................... 67
Introduction................................... 68
Materials and Methods.......................... 68
Results........................................ 71
Discussion..................................... 80

CHAPTER 4. Effect Of Pinealectomy on Prolactin,
Testosterone and Luteinizing Hormone
Concentrations in Plasma of Bull Calves
Exposed to 8 or 16 Hours of Light per Day. 83

Introduction................................... 84
Materials and Methods.......................... 85
Results........................................ 87
Discussion..................................... 98

CHAPTER 5. Melatonin and Prolactin Concentrations
in Serum of Bull Calves Exposed to 8, 16
or 24 Hours of Daily Light................ 103
Introduction................................... 104
Materials and Methods.......................... 105
Results........................................ 107
Discussion..................................... 123

SUMMARY AND CONCLUSIONS...0.0....OOOOOOOOOOOOOOOOOOOOOO 128
APPENDIXOIOOOO0......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 131

BIBLIOGRAPHY...0......OOOOOOOOOOOOOOOOOOOOOO0.0.00.0... 134

iii

List of Tables

Table 1. Pituitary Catecholamine Concentrations....... 78
Table 2. PRL Release from Anterior Pituitary Explants. 79

Table 3. Concentrations of Melatonin in Different
Volumes of Bovine Serum...................... 88

iv

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

List of Figures

Page

Schematic diagram depicting neural

connections from the eye to pineal gland of
mammals. Legend: RHP = retinohypothalamic
proiections, SCN = suprachiasmatic nucleus,

PVN = paraventricular nucleus, MFB = medial
forebrain bundle, RF = reticular formation,

IML = intermediolateral cell column, SCG =
superior cervical ganglia, ICN = inferior
carotid nerve, NC = nervi conarri.......... 25

Metabolic pathway for melatonin synthesis.. 29

Prolactin in serum from prepubertal bulls
switched from 8 h of light per day (at wk _

0) to continuous (24 L) or 16 h of light per

day. Each Observation represents the mean

Of four bulls where blood samples were

collected for 26 h through a jugular

cannula. Standard error of the difference
between treatments was 27.9 ng/ml of serum

and for wk was 10.6 ng/ml of serum.......... 42

Prolactin in serum from prepubertal bulls
switched from 8 h of light per day (at wk 0)

to continuous (24 L) or maintained on 8L:16D.
Each observation represents the mean of four
bulls where blood samples were collected for

6 h through a jugular cannula. Standard

error of the difference between treatments

was 5.5 ng/ml of serum and for wk was 4.6

nq/ml of serum.............................. 44

Prolactin in serum from bull calves exposed
to 8 h of light per day from 3 d to 8 wk of
age and subsequently switched to 16 b of
light per d or maintained on 8L:160. Each
observation represents the mean of four bulls
where blood samples were collected at 30—min
intervals for 6 h through a iugular cannula.
Standard error of the difference for treat-

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

ments was 23.9 na/ml of serum and for wk of
age was 9.8 nq/ml of serum.................. 54

Luteinizing hormone in serum from bull calves
exposed to 8 h of light per day from 3 d to 8

wk of age and subsequently switched to 16 h

Of light per d or maintained on 8L:16D. Each
Observation represents the mean of four bulls
where blood samples were collected at 30-min
intervals for 6 h through a iuqular cannula.
Standard error of the difference for

treatments was .23 ng/ml of serum and for

wk of age was .19 nq/ml of serum............ 56

Testosterone in serum from bull calves exposed

to 8 h of light per day from 3 d to 8 wk of

age and subsequently switched to 16 h of light
per d or maintained on 8L:16D. Each

observation represents the mean of four bulls
where blood samples were collected at 30-min
intervals for 6 h through a iuqular cannula.
Standard error of the difference for treat-

ments was .23 nq/ml of serum and for wk of

age was .29 ng/ml of serum.................. 58

Prolactin in serum from calves exposed to 8 h
of light per day from 3 d to 8 wk of age and
subsequently switched to 16 h of light per
day. Each observation represents the mean of
four bulls where blood samples were collected
at 30-min intervals through a iugular cannula
for 6 h. Castrations were performed at 1.5
to 2 wk of age. Standard error of the
difference for treatments was 14.1 ng/ml of
serum and for wk of age was 11.1 nq/ml of

Serum..0...0.000COOOOOOOOOOOOOOOOOCO0.0...O. 61

Dopamine in plasma from prepubertal bulls
receiving 8 or 16 h of light per day.

Each observation represents the mean of four
bulls where blood samples were collected for

6 h through a iugular cannula............... 72

Norepinephrine in plasma from prepubertal

bulls receiving 8 or 16 h of light per day.

Each observation represents the mean of four
bulls where blood samples were collected for

6 h through a iugular cannula............... 74

vi

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

Epinephrine in plasma from prepubertal bulls
receiving 8 or 16 h of light per day. Each
observation represents the mean of four bulls
where blood samples were collected for 6 h
through a jugular cannula................... 76

Twenty-six h profiles of melatonin in plasma
of bull calves receiving 16L:80 after 12 wk
on experiment. Each observation represents
the mean of four bulls. Samples were col-
lected at l-h intervals through a jugular
cannula. The hatched bar along the x-axis
represents darkness. Standard errors of
means during light and darkness; respect-
ively, were 6.8 and 21.8 pg/ml of plasma
for SPX calves and 6.3 and 6.0 for PX

CaIVGSOOOOOOOOOOOOOOCOO...OOOOOOOOOOOOOOOOO. 90

Twenty-six h profiles of melatonin in plasma
of bull calves receiving 8L:16D after 12 wk
on experiment. Each observation represents
the mean of four (SPX), three (Incomplete PX)
or one (PX) bulls. Samples were collected at
l-h intervals through a jugular cannula.
Hatched bars along the x-axis represent dark-
ness. Standard errors of means during light
and darkness; respectively, were 8.3 and 35.6
pg/ml of plasma for SPX calves, 6.9 and 27.2
pg/ml of plasma for incomplete PX calves and
6.4 and 5.4 pg/ml of plasma for the PX

ca].f.COO...0....0.00.0003...OOOOOOOOOCOOOOOO 92

Prolactin in plasma from PX and SPX bull
calves exposed to 8 h of light per day (at
wk 0) and subsequently switched to 16 h of
light per day or maintained on 8L:16D. Each
Observation represents the mean of four

bulls except the treatment combination of
8L:16D-PX which represents one bull. Samples
were collected at 30-min intervals for 26 h
through a jugular cannula. Standard error
difference for photoperiod treatment was

44.0 ng/ml of serum and for age was 27.8 ng/
ml of serum................................. 94

Age-adjusted testosterone in plasma from PX

and SPX bull calves. Each observation re-
presents the mean of eight (SPX) or five

vii

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

(PX) bulls where samples were collected at
30-min intervals for 26 h through a jugular
cannula . Standard error difference for sur-
gical treatment was .27 ng/ml of serum and
for age was .13 ng/ml of serum..............

Forty-eight h profiles of melatonin in serum
of four bull calves receiving 16L:8D after 4
wk on experiment. Samples were collected
through a jugular cannula at 30-min intervals
for the first 24 h and at 2 h intervals
thereafter. The hatched bar along the x-
axis represents darkness. Standard errors
of means during light and darkness; respect—
ively, were 8.2 and 27.3 pg/ml of serum.....

Forty-eight h profiles of melatonin in serUm
of three bull calves receiving 8L:16D after

4 wk on experiment. Samples were collected
through a jugular cannula at 30-min intervals
for the first 24 h and at 2 h intervals
thereafter. The hatched bar along the x-
axis represents darkness. Standard errors
of means during light and darkness: respect-
ively, were 6.6 and 18.0 pg/ml of serum.....

Twenty-four h profiles of melatonin in serum
of two bull calves receiving 8L:160. Samples
were collected at 30-min intervals through
a jugular cannula. The hatched bar along
the x-axis represents darkness. Standard
errors of means during light and darkness:
respectively, were 2.5 and 42.0 pO/ml of
serum for calf 8163 and 7.4 and 19.6 pg/ml
for calf 8133...............................

Twenty-four h profiles of melatonin in serum
of three (8164, 8170, 8171) or one (8166)
bull calves receiving 24L:0D. Samples were
collected at 30-min intervals through a
jugular cannula. Standard errors of means
were 1.6 pQ/ml of serum for calves 8164,

8170 and 8171 and 12.7 pg/ml for calf 8166..

Twenty-four h profiles of melatonin in serum
of three bull calves receiving 24L:0D and
infused intravenously with melatonin for 8 h
(hatched bar). Standard errors of means

viii

97

109

111

114

117

Figure 21.

during non-infusion were 26.6 pg/ml of
serum and 318.9 pg/ml during infusion....... 119

Twenty-four h profiles of melatonin in serum

of three bull calves receiving 24L:0D and

infused intravenously with melatonin for 16 h
(hatched bar). Standard errors of means

during non—infusion were 6.9 DG/ml of

serum and 141.8 pg/ml during infusion....... 121

ix

I NTRODUCTION

Researchers in animal science generally follow two
basic ideologies in terms of their rationale for conducting
research. The first is to gain knowledge which can
immediately benefit animal health and production and the
second is to gain knowledge for the sake of knowledge. I
chose to deal primarily with the latter aspect.

The focus of this thesis pertains to the effects of
daily duration of light (photoperiod) on endocrine function.
Many have demonstrated the benefits of photoperiod on
increasing milk yield in cattle (Peters et a1., 1978, 1981;
Stanisiewski et al., 1985), growth rates in cattle
(Petitclerc et al., 1983a: Tucker et al., 1984) and sheep
(Schanbacher and Crouse, 1981) as well as shortening the
duration from birth to puberty in cattle (Hansen et al.,
1983, Petitclerc et al., 1983a) and in swine (Mahone et al.,
1979, Ntunde et al., 1979). Yet, very little is known about
the mechanism through which photoperiod affects these
parameters; particularly, in cattle. Photoperiodic hormone
regulation is achieved through the pineal gland and its'
secretions in birds, hamsters and sheep. Whether this is

also true in cattle is unknown.

At present, it appears the popular trend among
consumers of animal products is to demand a healthful
product free of additives such as exogenous hormones and
other growth promotants. By better understanding the
mechanism through which photoperiod affects the endocrine
milieu in cattle, researchers may someday have the
Capability to consistently increase production efficiency
and alter carcass and milk composition (less carcass fat and
milk fat), using an acceptable agent to the consumer

(light).

Review of Literature

 

A. INTRODUCTION

It has long been known that season of the year as well
as daily duration of light exposure (photoperiod) can effect
several physiological parameters in mammals including
reproductive cycles, growth rates and lactational
performance (Wylie, 1925: Marshall, 1937: Peters et al.,
1978, 1981; Forbes, 1982). Manipulation of such parameters
using photoperiod can be an economical tool for the animal
industry to increase performance and efficiency of domestic
livestock. It is the purpose of this review of literature
to examine several biological factors which can be altered
with photoperiod. The factors I selected are by no means
all inclusive, but were selected for their potential (as
judged by myself) for economic benefit. Specific
introductions are given preceding each chapter of
experiments, however, this overall review of literature
should provide useful background information which is
intended to embellish the justification for following
certain protocols.
B. SEASONAL AND PHOTOPFRIODIC INDUCED CHANGES IN

HORMONES OF THE ANTERIOR PITUITARY

1 . PROLACTIN

Of the known non-gonadotropic, hypophyseal hormones,
prolactin (PRL) is most affected by season of the year in
all mammals in which it has been measured including seasonal
as well as non-seasonal breeders. Concentrations of PRL in
blood are 2 to 4 fold greater during the summer months in
cattle (Schams, 1972: Kensinqer et al., 1979: Tucker, 1982),
goats (Buttle, 1974: Muduuli et al., 1979: Howland et al.,
1985), sheep (Rhind et al., 1980: Kennaway et al., 1981),and
deer (VanMourik and Stelmasiak, 1985: Webster and Harrell,
1985) than in blood collected during winter months.
Photoperiod is one component of season which follows a
consistent pattern from year to year whereas, ambient
temperature tends to be more variable. Within limits, both
duration of daily light exposure and ambient temperature are
positively correlated with concentrations of PRL in serum of
cattle (Wettemann and Tucker, 1974: Leining et al., 1979),
goats (Muduuli et al., 1979) and sheep (Pelletier, 1973).
Several researchers have attempted to separate the effects
of photoperiod and ambient temperature on PRL concentrations
in blood. In heifers exoosed to a constant photoperiod of
12 h light: 12 h dark (12L:12D), increasing ambient
temperature from 21 to 32 C results in a linear increase in

l

PRL (1.2 ng/ml'C_ ): whereas, decreasing temperature from 21

C to 4.5 C results in decreased PRL (.6 ng/ml'C—l: Tucker
and Wettemann, 1976). A similar study using steers resulted

in a 4.2 ng/ml'C-1 increase in PRL when ambient temperature

increased from 20 to 40 C while reducing temperatures from

1 decrease (Smith et

20 C to 4 C resulted in a 1.85 ng/ml'C-
a1., 1977). Animal variation may account for the absolute
response differences of PRL to ambient temperature but
temperature appears to control serum PRL concentrations on a
minute to minute basis.

On the other hand, PRL responses to photoperiod tend to
be more sluggish. For instance, it takes 4 to 6 wk for PRL
concentrations in sera to increase in bull calves when
photoperiod is switched from 8L:16D to 16L:8D (Stanisiewski
et al., 1984). Similar response patterns are seen in rams
(Lincoln et al., 1978). Given the positive correlation
between temperature and day length on PRL release, it is
possible to account (using covariance analysis) for the
effects of each factor in animals maintained under natural
environment (seasonal) conditions. Temperature and
photoperiod account for most of the seasonal pattern of PRL
secretion in cattle, however, a small percentage of the
seasonal variation may be attributable to an endogenous
rhythm of secretion or to other unknown environmental cues
(Petitclerc et al., 1983b).

2. GONADOTROPINS

Other hormones of the anterior pituitary gland are
subject to regulation by season and photoperiod in some
species. Most notable are changes in gonadotropin secretion
associated with onset of reproductive activity in seasonally

breeding species such as goats, sheep and horses. Most

sheep and goats are reproductively quiescent during the non-
breeding season of spring/summer. But, in response to
reduction in day length , or to abruptly decreasing day
length from 16 to 8 h per day, plasma luteinizing hormone
(LH) and follicle stimulating hormone (FSH) begin an initial
increase within 2 to 4 wk after a photoperiod switch, with
peak responses occurring 1 to 5 wk later, again
demonstrating a sluggish hormonal response to photoperiod
(Lincoln and Davidson, 1977). Peak endogenous pulses of LH
and FSH are 3-and 10-fold greater: respectively, when
compared with the quiescent period in rams. Likewise, LH in
serum of ewes are approximately 12-fold (6 vs .5 ng/ml)
greater during the breeding season than during the non-
breeding season (Bittman et a1., 1983). Pulsatile activity
of LH increases from 3.6 to 5.4 pulses/24h in rams during
the breeding season when compared with the non-breeding
season (Schanbacher and Ford, 1976) or from 1 to 10
pulses/24h when comparing long days with short days (Lincoln
et a1., 1977). LH concentrations in another short day
breeder, the pygmy goat, follow a seasonal pattern similar
to that seen in sheep (Muduuli et al., 1979: Howland et al.,
1985). Reproductive activity ceases in short-day breeders
in response to increasing day lengths.

In contrast, horses are long-day breeders, and
increasing duration of daily light from 9 to 16 h induces
100 to 400 fold increases in serum LH in mares within 7 to

14 wk (Oxender et al., 1977). Seasonal changes in

cirCulating gonadotropin concentrations are positively
correlated with pituitary gonadotropin content indicating
that changes in hormone synthesis rates are probably
occurring in the brain (Berndtson and Desjardins, 1974). In
cattle, a non-seasonal breeder, LH and FSH are unaffected by
photoperiod (Rzepkowski et al., 1982).

3. STEROID HORMONES

In general, gonadal steroid release is under direct
control of the gonadotropins, therefore, reproductive
steroids tend to follow a similar circannual pattern to the
gonadotropins in seasonally breeding mammals. Breeding
season induced rises in LH and FSH are followed immediately
by increased testosterone or estradiol concentrations in
serum of rams, (Lincoln and Davidson, 1977) ewes (Legan and
Karsch, 1979) and pygmy goats (Muduuli
et al., 1979). Progesterone and estradiol serum
concentrations are greater during spring/summer months in
mares, and can be elevated during winter months using a
16L:8D photoperiod (Oxender et al., 1977). Furthermore,
serum testosterone in stallions is greatest in May (3.2
ng/ml) and lowest during December (1.5 ng/ml) and is
correlated with the normal spring/summer breeding season
(Berndtson et al., 1974). An important sex difference
exists however, in that the hypothalamo-hypophyseal-ovarian
axis remains functional (though anovulatory) in ewes during
the anestrous season which is in contrast to rams in which

gonadal regression takes place. Changes in feedback

sensitivity are perhaps responsible for the ewes inability
to demonstrate preovulatory LH surges under long days. In
fact, anestrus in the ewe may be due to‘a long-day induced
increase in sensitivity to estradiOl negative feedback on LH
secretion (Legan and Karsch, 1979).

Lincoln et a1. (1977) demonstrated circadian changes in
testosterone concentrations in sexually active rams with
highest concentrations tending to occur at night (between
1700 and 2000b). This was attributable to increased
frequency of gonadotropin pulses occurring at night, a
phenomenon which has also been described in men (Boyer et
al., 1972) and rats (terHaar et a1., 1974).

Glucocorticords do not appear to vary with any seasonal
or photoperiod-induced rhythmicity in cattle (Leining et
al., 1980), white-tailed deer (Bubenik et al., 1975) Or rams
(Lincoln et a1., 1982). However, in male pygmy goats,
cortisol concentrations in serum change with season and at
times are directly correlated with pulsatile changes in
testosterone (Howland et al., 1985).

C. HYPOTHALAMIC CONTROL OF PROLACTIN SECRETION

PRL is secreted episodically into blood of most species
including cattle (Stanisiewski et al., 1984). Furthermore,
PRL increases in blood within a few minutes by stimuli such
as stress (Johke, 1970: Dunn et a1., 1972) and rising
ambient temperature (Tucker and Wettemann, 1976). In
addition, PRL release is controlled by less acute means,

such as in response to changing day lengths.

Several hypothalamic PRL-release (PRF) and-release
inhibiting factors (PIF) are presumed to directly control
PRL synthesis and (or) release from the anterior pituitary
gland. For instance, following lesioning of the median
eminence in rats, PRL increases rapidly in plasma and
elevations are maintained, presumably in response to acute
release of PRF from lesioning plus chronic removal of PIF
(Bishop et al., 1972). Furthermore, rat hypothalamic
extracts inhibit pituitary PRL release in vitro (Shaar and
Clemens, 1974).

Hypothalamic catecholamines may be responsible for
physiological control of PRL release. For example, dopamine
(DA) inhibits PRL release in vitro (Shaar and Clemens, 1974:
Padmanabhan et a1., 1979) while epinephrine (EPI) and
norepinephrine (NE) inhibit (Birge et al., 1970; Shaar and
Clemens, 1974), increase (Koch et a1., 1970) or have no
effect (Talwalker et al., 1963) on PRL release. Therefore,
it appears that EPI and NE may have some control of PRL
release, but the predominant PIF appears to be DA (Leong et
a1., 1983). In fact, cells of.the anterior pituitary gland
(presumably lactotrophs) have the capacity to internalize
and process DA (Gudelsky et al., 1980).

There are, no doubt, several additional factors such as
thyrotropin-releasing hormone (TRH), serotonin, and
vasoactive intestinal peptide, that may control PRL
secretion (Leong et a1., 1983). In fact, Wolinska et al.

(1977) have shown in sheep that lesions to the anterior

medial basal hypothalamus (MBH) decrease plasma PRL:
whereas, caudal MBH lesions increase PRL. It appears from
these data that at least two functional systems are at work
in the hypothalamus which stimulate or inhibit PRL release.
Perhaps the most prevalent hypothesis concerning PRL release
suggests that decreased DA inhibition depletes a releasable
pool of PRL from the anterior pituitary gland. Then, a
second intracellular pool of PRL becomes releasable by TRH
(Grosvenor and Mena, 1980: Lafond and Collu, 1986).
Similarly to DA, TRH acts directly on the pituitary gland to
regulate PRL release (Clemens et a1., 1980).

It is unknown how photoperiod may affect hypothalamic
factors which in turn alter PRL secretion. However, a
likely candidate for study would be DA, since it appears to
be responsible for tonic PRL inhibition. In addition, Naber
et a1. (1981) have shown that rats possess an endogenous
rhythm in DA receptor binding which undergoes seasonal
variations. In addition, humans have reduced concentrations
of DA in hypothalami during spring/summer compared with
fall/winter (Karson et a1., 1984). Finally, DA and its
metabolites are higher in the retina of rats during periods
of light as compared with darkness (Cohen et a1., 1983:
Melamed et a1., 1984), suggesting further that photoperiodic
influences on PRL may be partially mediated through DA.

D. SEASONAL AND PITUITARY CONTROL OF GONADAL FUNCTION
Many animals have evolved into seasonal breeders with

the primary function being that reproduction occurs at a

10

time of the year when Offspring can be reared in a favorable
environment, thus allowing for maximum survival. Although
many environmental factors change with the seasons,
photoperiod is the primary cue governing reproductive
activity or quiescence. Ultimately, the most advantageous
time of the year to reproduce is determined by food supply,
predation and climate (Baker, 1938): however, photoperiod is
used as an advanced announcement of a change in environment.
Some species become reproductively active in response to
long days (eg. hamsters, ferrets, horses) while reproductive
activity is induced in other species by short days (eg.
sheep, goats, deer, mink :Turek and Campbell, 1979: Tucker
and Oxender, 1980). Still other animals (eg. cattle,
domestic rabbit) are reproductively active without regard to
photoperiod. To complicate the situation further, exposing
animals to a constantly repetitive photoperiod which induces
a characteristic hormonal or reproductive response will not
maintain that response indefinitely. For example, PRL
concentrations in serum and pituitary glands decrease in
hamsters exposed to short days (Bex et al., 1978: Reiter et
a1., 1975). However, PRL in blood increase spontaneously
even when short days are maintained (Goldman et al., 1981).
In addition, gonadal regression takes place in hamsters
exposed to short days (Steger et al., 1985), but spontaneous
gonadal recrudescence occurs within 10 wk of maintained
short days (Gaston and Menaker, 1967). When hormone

secretion or gonadal responses overcome the applied

11

photostimulation, the animals are said to have become
refractory (Farner and Follett, 1966). Photorefractoriness
has also been documented in sheep (Robinson et a1., 1985)
and several species of birds (Follett, 1978). Therefore,
the neuroendocrine mechanism controlling gonadal function in
seasonal breeders is not just passively following seasonal
photoperiod changes. In actuality, the internal biological
clock which animals are purported to have, appears to be
running on its own endogenous rhythm and is likely being
cued and reset by changing photoperiods.

As pointed out previously, photoperiod-induced changes
in gonadal steroid concentrations are fairly well correlated
with gonadotropin concentrations. In the classical sense,
gonadal steroid concentrations have a negative feedback
effect on gonadotropins (Nalbandov, 1976). However,
photoperiodic species appear to be an exception to this
rule, at least during certain seasons of the year. In fact,
exogenous steroids given in conjunction with a stimulatory
photoperiod (breeding season) is a less potent inhibitor of
gonadotropins than when given during a non-stimulatory
photoperiod in sheep (Pelletier and Ortavant, 1975: Legan
and Karsch, 1979) and hamsters (Tamarkin et al., 1976: Ellis
and Turek, 1979).

In spite of the changes in circulating hormones taking
place in response to photoperiod, very little is known about
the direct effects of these changes on the gonad. LH and

FSH are considered to be essential for maintaining

12

reproductive function in mammals of both sexes (Sanford et
a1., 1978), whereas the reproductive role of PRL is less
well defined except for its luteotrophic effect in certain
rodents (Murphy and Raikumar, 1985) and perhaps sheep
(Denamur et al., 1973). In addition, Rhind et a1. (1980)
established that PRL concentrations in blood during estrus
are greater in sheep during March or July (normal anestrus)
versus December (normal breeding season). These authors
suggest an anti-fertility role for PRL in ewes. Still other
evidence suggests that PRL may play a mediating role between
photoperiod and feedback sensitivity (Matt et al., 1984).
Testosterone is more effective in suppressing LH and FSH in
hamsters soon after switching from a short to a long-day
photoperiod when compared with suppression observed 30 days
after the switch, and this effect is more marked in
dopamine-agonist treated hamsters, indicating that decreases
in PRL increase feedback sensitivity (Matt at al., 1984).
Estrogens and aromatizable androgens administered to
castrated rams increase PRL concentrations in serum (Parrott
and Hill, 1979) and additional evidence indicates that
testosterone inhibits PRL release in wethers exposed to
8L:16D (Pelletier, 1973). Although the evidence is
conflicting, suffice it to say that the gonadal steroids may
in part modulate their own seasonal secretion via a direct
effect on PRL.

In all species in which it has been examined, LH has a

direct effect on testosterone production by the gonad

13

(Bartke et a1., 1978). The androgen stimulating action of
LH appears to be almost exclusively on the Leydig cells
where the mechanism involves synthesis of proteins as well
as increased activity of enzymes involved in testosterone
production.
E. PUBERTY
1 . DEFINITIONS
Defining puberty is often an obscure proposition,
particularly in the case of males. However, the following
is a list of attempts from a variety of sources.
"The period in both males and females marked by the
functional development of the generative system".
Webster's Dictionary (1975).
"Pituitary growth and its hormonal influence on body
growth and ovarian activity stimulate growth and development
of the uterus and other parts of the reproductive tract.
These changes result in the reproductive tract of the heifer
becoming functionally Operative. Thus puberty is reached."
Salisbury et a1. (1978).
"Puberty is the transitional period between the
juvenile state and adulthood during which the adolescent
growth spurt occurs, secondary sexual characteristics
appear, fertility is achieved, and profound psychological
changes take place."
Reiter and Grumbach, (1982).
"Puberty might be defined as the point in development
when a bull is capable of participating in reproduction."

Amann and Walker, (1983).
14

There are probably as many variations on the definition
of puberty as there are people to give it a definition.
However, it is not my concern to add to this list, although
for the sake of continuity of this review, I shall refer to
puberty as the achievement of sexual maturation and the
ability to reproduce. However, my task is to examine the
endocrine and neuroendocrine parameters which lead to
puberty and how these parameters can be altered.
Furthermore, I will limit this discussion primarily to males
with some consideration of females where appropriate. The
prepubertal sequence is a complex series of events, and in
bulls is thought to contain the following order:
l)initiation of spontaneous discharge of LH, 2)hormone
induced differentiation of Leydig cells with increased
secretion of androstenedione in response to LH stimulation,
3)further differentiation of Leydig cells resulting in LH-
stimulated secretion of testosterone, 4)testosterone-induced
differentiation of indifferent supporting cells to Sertoli
cells concomitant with testosterone-induced differentiation
of gonocytes to prespermatogonia and A-spermatogonia,
5)increased sensitivity of the hypothalamus-anterior
pituitary to negative feedback of gonadal steroids,
6ldiminished frequency and amplitude of LH discharge, 7)
formation of functional complexes between Sertoli cells and
establishment of the blood-testis barrier, 8)formation of
primary spermatocytes and ultimately spermatids and

spermatozoa, and 9)continued increase in efficiency of

15

spermatogensis until sufficient sperm are produced to
provide the first ejaculum around 37 to 38 wk of age (Amann,
1983). In the following sections, I will review some of the
hormonal changes taking place during the prepubertal period
and examine how some of these changes may be altered.

2. ENDOCRINE CHANGES ASSOCIATED WITH ONSET OF PUBERTY

In mammals, secondary sex characteristics as well as
the capacity to reproduce are under direct control of
gonadal steroids. However, production of gonadal steroids
are dependent upon secretions of the anterior pituitary
gland (Bartke et al., 1978). For example, several reports
indicate that circulating LH concentrations in bulls begin a
gradual increase between 1 and 5 mo of age (Rawlings et al.,
1978: McCarthy et al., 1979a, 1979b: Amann and Walker, 1983)
and may in fact increase further up to 10 mo of age
(MacMillan and Hafs, 1968: Rawlings et al., 1978).
Testosterone in blood likewise increases gradually from 1 to
10 mo of age but the rate of increase is most pronounced
around 4 to 6 mo (Karg et al., 1976: Bedair and Thibier,
1979: McCarthy et al., 1979b). However, highest
concentrations of testosterone generally lag behind
increased LH concentrations by about 1 mo (McCarthy et al.,
1979a). In contrast, age related differences in FSH have
not been detected (McCarthy et al., 1979a, 1979b) except in
one case in which FSH in blood increased a modest 30%
between 1 and 8 mo (Amann and Walker, 1983). Similarly to

calves, rams also have a well defined prepubertal period

16

where LH concentrations increase initially around 5 wk of
age, then exhibit a secondary rise by wk 41 before declining
(Lee et al., 1976). Likewise, FSH concentrations in sheep
are maximal by 5 wk and maintained at those levels while
concentrations of testosterone are closely correlated with
LH.

Prepubertal hormone changes are relatively well
established in rats. In fact, LH concentrations in blood of
rats plateau around d 10 while FSH concentrations are
maximal around d 30 then decline (Payne et al., 1977). The
decline of FSH concentrations coincide with development of
mature spermatogenesis (Swerdloff et al., 1971). Whether
these gonadotropin concentrations in rats correspond to a
prepubertal period similar to that of large domestic animals
is in question since spermatogenesis in rats is reported to
begin around the time of birth (Lee et al., 1976).

Reports conflict for sheep and cattle in terms Of age
related changes in circulating PRL, ranging from no age
effects (Schams and Reinhardt, 1974) to increased PRL
concentrations at 3 mo (Leining et al., 1976: Davis et al.,
1977) or 4 to 5 mo of age (McCarthy et al., 1979a). Ravault
and Courot (1975) reported a marked increase of PRL
secretion in male lambs coincident with rapid growth of
testis and establishment of spermatogenesis. At present
however, there is no good evidence linking PRL secretion
with puberty onset in large domestic animals (i.e. sheep and

cattle) at least in terms of spermatogenesis, testis weight

17

or L8 and testosterone secretion. However, PRL deficiency
results in reduced weight of seminal vesicles in lambs
(Ravault et al., 1977: Ohlson et al., 1981). On the other
hand, PRL in rats increases gradually through the time of
puberty (Dohler and Wuttler, 1974, 1975) and in male rats
PRL is reported to have a synergistic effect with LH on
spermioqenesis (Bartke, 1971), and testosterone production
(Hafiez et al., 1972). Furthermore, PRL synergizes with
testosterone to stimulate growth of accessory sex glands in
male rats (Moger et a1, 1972: Hostetter and Piacsek, 1977).

3. NEUROENDOCRINE EFFECTS ON PUBERTY

There are three principal components to the control
mechanism of puberty onset in mammals (Reiter and Grumbach,
1982). First is the arcuate nucleus of the medial basal
hypothalamus along with its projections. Gonadotropin-
releasing hormone (GnRH) is synthesized and released by
these secretory neurons into the median eminence where GnRH
is transported via hypothalamic-hypophyseal portal
circulation to the second component of the system, the
pituitary gonadotropes. Gonadotropes release LH and FSH in
a pulsatile manner in response to the rhythmic GnRH signal.
Finally, LH and FSH directly affect the gonads. I

Most data suggest that the major control point of the
system is at the level of the hypothalamus. For instance,
GnRH is detectable in fetal human hypothalami by 10 wk of
gestation (Kaplan et al., 1976), and in rats as early as d

17 of gestation (Chiappa and Fink, 1977). Furthermore, in

18

humans (Groom and Boyns, 1973) and rats (Root et al., 1975)
prenatal and neonatal pituitary gland fragments are capable
of LH and FSH release in response to GnRH. George et al.
(1979) have shown that in rabbit fetuses testosterone
secretion becomes gonadotropin dependent beginning in late
gestation. Therefore, most controlling influences on
puberty onset occur at the hypothalamic level, where
initiation Of the reproductive process likely begins.
Several studies suggest that the central nervous system
(CNS) has a major impact on puberty onset, namely through a
mechanism restraining onset of puberty (Reiter and Grumback,
1982). This restraint is removed as puberty approaches
(Ramaley, 1979) and has been commonly referred to as the
gonadostat theory of puberty onset (Raimirez and McCann,
1965). CNS control of puberty onset probably acts through
brain neurotransmitters, such as the catecholamines. For
instance, inhibition of catecholamine synthesis in 20-d old
rats abolishes hemiovariectomy—induced ovarian hypertrophy
(Muller et al., 1972). Those same authors concluded that
norepinephrine (NE) was the primary catecholamine involved
with prepubertal FSH release. In addition, there is a
marked increase in hypothalamic NE turnover preceding
prepubertal LH increases in rats (Advis et a1., 1978: Wuttke
et a1., 1980). Likewise, in rats, the usual castration-
induced rise in LH and FSH secretion can be inhibited by
blocking NE synthesis (Cocchi et al., 1974). Wuttke et al.

(1980) reported an increase in hypothalamic NE and DA

19

turnover between d 21 and puberty in rats while others
showed decreased DA and increased turnover of serotonin
preceding the first LH surge in rats (Advis et al., 1978).
Advis et a1. (1978) claimed the changes in DA and serotonin
could be responsible for prepubertal increases in
circulating PRL of rats.

4. MANIPULATION OF PUBERTY ONSET

Several factors can be used to manipulate onset of
sexual maturity in animals and one which certainly has a
marked effect is level of feeding. For example, dairy
heifers with restricted feed intakes reach sexual maturity
later than full-fed controls (Sorensen et al., 1959). These
data would suggest that body size is more important than age
in determining sexual maturity. However, I will not dwell
further on factors which are restrictive in nature, but
rather will examine situations where environment and or
endocrine manipulation hasten puberty.

Photoperiod has been used extensively to alter sexual
maturation in a variety of species. Much of the work on
photoperiod-induced puberty onset has been accomplished in
rodents. For example, short-day photoperiod exposure from
birth delays gonadal development in the mouse, cotton rat,
field vole and Diungarian hamster (Lecyk, 1962: Hoffmann,
1978: Johnston and Zucker, 1979: Zucker et a1., 1980). Many
of the animals in which photoperiod affects puberty onset
are seasonal breeders. That is, photoperiod controls

reproduction throughout their reproductively active life.

20

Therefore, one might expect photic influences to be
important during reproductive development. Sexual
maturation, however, is unaffected by photoperiod in some
seasonal breeders such as the Syrian hamster (Gaston and
Menaker, 1967: Darrow et al., 1980).

As might be expected, photoperiodic effects on sexual
maturation are closely associated with many endocrine
changes. For example, Djungarian hamsters grown under long
days have increased FSH, LH, PRL and androgen concentrations
in serum between 15 and 30 d of age in addition to increased
body and testis weights compared with animals receiving
short days (Yellon and Goldman, 1984). However, Djungarian
hamsters maintained under short days eventually become
refractory to this inhibition and attain full gonadal
function by 150 d of age (Hoffmann, 1978). In addition,
gonadal response to photoperiod in neonatal hamsters may
also be dependent on maternal photoperiod exposure, and
transfer of that information (Stetson et al., 1986). For
example, gonadal maturation rates of offspring are hastened
when gestated with 16 h compared with 12 h of daily light.

As stated earlier, both age and body weight are
determinates of when puberty occurs, and weight is more
important. A long-day photoperiod (18L:6D) will reduce both
age and body weight at puberty in female rabbits compared
with 6L:18D exposure (Kamwanja and Hauser, 1983)
Furthermore, gilts born during the autumn and maintained on

natural photoperiod (day lengths are decreasing) attain

21

puberty at a younger age and lower weight than gilts born in
the spring (Wiggins et al., 1950: Mavrogenis and Robison,
1976). In contrast, exposing boars (Mahone et al., 1979:
Berger et al., 1980) and gilts (Ntunde et a1., 1979) to 15
to 18 h of light daily accelerates onset of puberty relative
to animals under natural winter photoperiods (9 to 12.7 h of
daily light). However, Ntunde et al. (1979) could not
attribute the photoperiod effect on puberty to alterations
in circulating LH concentrations. The reason natural and
artificial photoperiods appear to affect swine differently
is unclear.

Similar to swine, in cattle season and photoperiod can
also affect timing of puberty onset. For example, heifers
born in winter or spring were 52 d younger and 54 kg lighter
at puberty than similar heifers born during summer or fall
(Roy et al., 1980: Little et a1., 1981). In addition,
16L:8D hastens puberty onset and body weight gain in heifers
compared with animals receiving 8 b of daily light
(Petitclerc et al., 1983a). Hansen et a1. (1983) also
reduced age at first ovulation in heifers with 18 h of daily
light compared with natural autumn day lengths but, as with
swine, L8 in serum was unaffected by photoperiod. Effects
of photoperiod on puberty onset in bulls has not been
determined. However, in one of the first experiments to
examine photoperiod effects on puberty in rams, Howles et
al. (1980) saw no difference in testosterone concentrations

or testis growth in response to 8 or 16 h of light per day.

22

F. LIGHT PERCEPTION AND ENDOCRINE CHANGES

1. FUNCTIONAL ANATOMY

It has long been known that seasonality and photoperiod
can alter diurnal as well as circannual activity rhythms of
a variety of mammals. However, the mechanism through which
light and (or) dark impact upon the animal is generally not
understood. Ouay (1956, 1963) was one of the first to
demonstrate that both structure and function of the
mammalian pineal gland could be altered by photoperiod.
Around the same time, Lerner et al. (1960) isolated the
indolamine, melatonin, from bovine pineal tissue while
Wurtman et al. (1963) showed that melatonin synthesis was
controlled by light. Thus, the foundation of the photic-
endocrine mechanism was in place, but further studies were
needed to establish the precise relationship of the pineal
gland with rhythmicity in animals. The following is a
review describing the neural connections between the eyes
and the pineal gland in mammals. Most of the anatomical
relationships have been determined in rats.

In terms of photic perception and light measurement,
mammals are at a disadvantage compared with non-mammalian
vetebrates. Light can pass through the relatively thin
skulls of birds and impinge directly upon the pineal gland
(Underwood and Groos, 1982). Mammals on the other hand,
being thick-skulled creatures, had to develop an alternate
pathway to permit light signals to reach the pineal gland.

This alternate system involves an external photoreceptor

23

(eye), which transmits lightzdark signals via a complex
sympathetic network to the pineal gland (figure 1). The
first links in the network are retinohypothalamic neural
projections from the retina primarily to the contralateral
suprachiasmatic nucleus (SCN) within the hypothalamus
(Moore, 1983). The SCN is commonly referred to as the
master oscillator or pacemaker controlling many circadian
rhythms (Klein et al., 1983: Moore, 1983: Turek, 1985).
Indeed, it was discovered that destruction of the SCN or its
neural connections results in disruption of circadian
rhythms in rodents (Turek, 1983). Although the SCN varies
in size and shape among mammals, they are always laterally
symmetrical and located near the third ventricle and dorsal
to the optic chiasm (Turek, 1985). Furthermore, the SCN
develops gradually after birth and in the rat does not
acquire full adult organization and features until 5 wk of
age (Lenn et al., 1977). Axons of SCN neurons project to
the paraventricular nucleus (PVN) and perhaps synaptic
contacts are made within the PVN (Klein et al., 1983).‘ In
fact, complete destruction of either the‘SCN or PVN is
required to block photic stimulation of the pineal gland in
rats or monkeys (Klein and Moore, 1979: Reppert et al.,
1981: Klein et al., 1983). For instance, as little as 5% of
the PVN is sufficient for normal circadian enzyme function
in the pineal gland (Klein et a1., 1983). Although further
neuronal connections to the pineal gland are less well

defined, several pathways have been hypothesized (Swanson

24

Figure 1.

Schematic diagram depicting neural
connections from the eye to pineal gland of
mammals. Legend: RHP = retinohypothalamic
projections, SCN = suprachiasmatic nucleus.
PVN = paraventricular nucleus, MFB = medial
forebrain bundle, RF = reticular formation.
IML = intermediolateral cell column, SCG =
superior cervical ganglia, ICN = inferior
carotid nerve, NC = nervi conarri.

Redrawn from: Moore and Klein, 1974.

Klein et al., 1983.
Reiter, 1983.

25

 

Eye

Pineal gland

 

     
  

Cerebrum NC
PVN

SCN

V
"__"y .P
- 8\
E e
Y T»

W

Sagittal View

ICN

 

 

 

 

 

Ventral View

26

scc'

IML

and Kuypers, 1980). The preponderance of information
suggests that axons project from the PVN through the medial
forebrain bundle (MFB) and reticular formation (RE) where
long descending axons carry the signal to the.
intermediolateral cell column Of the upper thoracic spinal
cord (Klein et al., 1983: Reiter, 1983). Preganglionic
sympathetic neurons then project up the sympathetic trunk
and terminate within synapses of the superior cervical
ganglia (SCG). The SCG are bilateral, and lie at the
junction between the common carotid and the internal and
external carotid arteries. Postganglionic axons proceed from
the SCG via the inferior carotid nerve (ICN) to the vicinity
of the pineal gland where they form distinct nerve bundles
(nervi conarri: Kappers, 1960). Neural projections then
penetrate the capsule of the pineal gland and terminate
primarily in pericapillary spaces (Reiter, 1983). Until
recently, it was thought that the mammalian pineal gland
acted exclusively as a neuroendocrine organ under
orchestration from efferent sympathetic innervation.
However, Korf et al. (1986) demonstrated in hamsters that
pinealocytes send neural projections to various areas of the
brain. Therefore, the mammalian pineal gland may
communicate directly via neurons with various areas of the
brain in addition to its neuroendocrine function.

2. PINEAL GLAND FUNCTION

Photoperiodic information from an animals' environment

is translated into a neural signal which travels the

27

circuitous pathway previously described between the eyes and
pineal gland. The pineal gland in turn functions as an
endocrine organ and converts the neural signal into a
hormonal signal. The primary secretory product of the
pineal gland is melatonin. Melatonin is a hormone which 35
secreted in a circadian pattern under regulation of the CNS
(Wurtman et al., 1964a).

3. MELATONIN SYNTHESIS AND SECRETION

Melatonin is synthesized from the amino acid,
tryptophan, by the synthetic indole pathway diagramed in
figure 2. There are three important points concerning this
pathway. First, of the two enzymes responsible for
converting serotonin to melatonin (N-acetyltransferase, NAT:
and hydroxyindole-O-methyltransferase, HIOMT), NAT-is rate-
1imiting for melatonin formation (Klein, 1973) and is'
frequently described in the literature as an index of
melatonin synthesis. HIOMT serves as a marker for specific
sites of melatonin formation. Second, essentially all of
the indoles are found in relatively high concentrations in
the pineal gland with serotonin attaining greatest
concentrations. Furthermore, serotonin turnover is greater
in pineal tissue than any other area of the CNS (Cardinali,
1981). The final point I would like to make is that the
pathway for indole synthesis occurs in tissues other than
the pineal gland including Harderian gland (Cardinaili and
Wurtman, 1972), retina (Cardinali and Resner, 1971) and

intenstine (Bubenik et al., 1977). However, except for

28

Figure 2. Metabolic pathway for melatonin synthesis.

Source: Martin et al. 1976.

29

COOH

H
/
H CH ’CH
' 2 \NHZ tryptophan
H N

H H
l tryptophan hydroxytase
H 0008
H0 CHZ'CH\
H N I H S-hydroxylryptophan
l-I

aromatic l-amino
acid decarboxyiase

H

CHz-CHZ-NHZ _ Serotonin
G-hyoroxytryptamine)

N-acetyltransferase

HO CH -CH -NH-c-c
“‘7" 2 2 H3 N-acetylserotonin

H

 

l: Hydroxylndole-O-methyltransferase
H

CHZ'CHz-NH'g'CH; Melatonin
:Hl (S-methoxy
N-acetylserotoninl

30

minor contributions From the retina (Hamm and Menaker, 1980:
Pang et al., 1980), diurnal variation of melatonin secretion
is attributed primarily to pineal tissue. Extra-pineal
melatonin sources are probably reSponsible for persistent
low concentrations of circulating melatonin after pineal
gland removal (Oiaki and Lynch, 1976: Kennaway et a1., 1977)
but function of this melatonin is unknown.

4. CHARACTERITICS OF MELATONINI

.M

. . f" .
Most studies confirm that the.@mount of melatonin

present in pineal tissuelis a direct reflection ogimelatonin
concentrations in blood, urine (Ozaki and Lynch, 1976) and
usually CSF?(Minneman and Wurtman, 1976: Wurtman and Hedlund
et al., 1977: Moskowitz, 1977). In addition, melatonin is
not stored for extended lengths of time within the pineal
gland, but is secreted as it is produced (Reiter, 1983).
The half-life of melatonin in blood of rats and sheep is
approximately 20-min (Cardinali et al., 1983) but may be
longer in neonatal rats due to lower catabolism of melatonin
by liver (Weinberg et al., 1981).

Radioactive melatonin iniected systemically is taken up
by virtually every tissue oF the body (Kopin et al., 1961).
Specific tissues such as the pineal gland, eye, gonads,
adrenal and hypothalamus appear to take up relatively high
concentrations of the hormone (Wurtman et al., 1964b; Anton-
Tay and Wurtman, 1969). Specific melatonin receptors have

since been Found in the medial basal hypothalamus (MBH)

(Cardinali et a1., 1978), pituitary gland (Lang and

31

Sizonenko, 1980), and in peripheral tissues such as gonads,
uterus, liver and eyes (Cohen et a1., 1978). Highest
affinity binding sites for melatonin seem to be in the HRH.
Actions of melatonin on peripheral tissues have not been
determined.

Several reports indicate that melatonin secretion
decreases with age (Reiter, 1983). Basal melatonin
concentrations (with lights on) do not appear to be affected
by age, but nocturnally produced melatonin is approximately
3-fold higher in 2—mo old than in l8-mo old hamsters (Reiter
et al., 1980). In addition, diminished nocturnal production
of melatonin with age is a gradual process in rodents
(Reiter et al., 1981a). In humans, peak nocturnal melatonin
concentrations increase from birth to about 5 yr of age, but
thereafter melatonin declines through puberty, plateaus, and
then declines further with old age (Reiter, 1986). The
prepubertal melatonin decline is suggested to be a trigger
for commencement of prepubertal events in children (Kolata,
1984).

5. BIOLOGICAL EFFECTS OF MEILATONIN

More melatonin is secreted from the pineal gland during
darkness than during periods of light in virtually all
species examined (Wurtman et a1., 1983). However, the
pattern of nocturnal melatonin varies among species (Reiter,
1983). For example, Syrian hamsters exhibited peak
melatonin secretion late in the dark period: whereas, albino

rats and Turkish hamsters show peak melatonin activity

32

during the middle of the dark period. In contrast, peak
melatonin concentrations appear in Richardson's ground
squirrel early in the dark period (Reiter et al., 1981b).
Another variation in melatonin secretory patterns appears in
Diungarian hamsters, cotton rats, white-footed mice (Reiter,
1983) and Suffolk sheep (Bittman et a1., 1983) where
nocturnal pattern of melatonin secretion closely corresponds
to the total duration of darkness provided.

The means by which melatonin conveys photic information
so that an animal can adiust its' endocrine profile in
accordance with season has only recently been examined. Two
maior hypotheses exist, which are not necessarily exclusive
(Reiter, 1986). The first hypothesis builds on the fact
that in some species, the longer the night, the more
prolonged is the daily melatonin rise. This is the duration
hypothesis, which stated simply, relays photic information
to an animal through duration of increased daily melatonin
secretion from the pineal gland. In fact, it is duration of
elevated melatonin concentrations which control reproductive
and hormonal responses to melatonin in sheep (Bittman and
Karsch, 1984) and Diungarian hamsters (Goldman et a1.,
1984). The second hypothesis, known as the internal
coincidence hypothesis, predicts the existence of two
rhythms. One rhythm is provided by diurnal melatonin
secretion and the second is an endogenous rhythm of
sensitivity to melatonin. The animal's physiological

response is based on the synchrony and coincidence of these

33

two rhythms (Reiter, 1986). Predictably, the internal
coincidence hypothesis is much more difficult to test,
particularly in terms of melatonin sensitivity.
G. OBJECTIVES

The overall obiective of the following series of
experiments is to study the effect of photoperiod on
hormones which may be important in photoperiod-regulated
growth and onset of puberty responses, and to examine the
mechanism through which photoperiod affects circulating
hormone concentrations in bull calves. The following
general design was utilized in five different series

(chapters) of experiments.

EXPERIMENTAL DESIGN:

 

 

 

 

 

 

 

 

 

LIGHT CONTROLLED LIGHT, TEMP. CONTROLLED
O t a
CHAMBER CHAMBER
BLIIGD I6 L3 8 0
8L: I60
amen 8L = I6 0
I
O WK 6WK BWK I4 WK ZOWK
BLOOD COLLECTED

34

Modifications of this design are explained within the text
of each chapter and consist primarily of differences in
photoperiod treatments and blood collection intervals.
Eight bull calves were placed into the light controlled
chamber within 3 d of age and assigned to one of two light-
and temperature-controlled chambers (four calves per

chamber) at approximately 6 wk of age.

35

Chapter 1

Effect of Continuous Light on Prolactin Secretion in

Prepubertal Holstein Bulls

36

Introduction

 

A light-dark cycle of 16 h light:8 h dark (16L:8D)
increases milk yield (Peters et al., 1978), growth rates
(Peters et al., 1978) and hastens puberty onset (Petitclerc
et al., 1983a) in cattle compared with 9 to 12 h daily
light. Prolactin (PRL) concentrations in blood of cattle
are consistently modulated by photoperiod. For example,
after abruptly switching from 8L:16D (short-day) to 16L:8D
(long-day), at least 1 wk elapses before concentrations of
PRL in serum begin to increase (Leining et a1., 1979:
Stanisiewski et al., 1984) and within 4 to 6 wk,
concentrations of PRL increase about S-fold in serum of bull
calves (Leining et al., 1979). Conversely, decreasing daily
duration of light exposure from 16 to 8 h decrease PRL
concentrations in serum (Bourne and Tucker, 1975). Similar
PRL changes in response to photoperiod have been reported
for sheep (Lincoln et al., 1978; Sanford et a1., 1978).

Rats exposed to 24L:OD have greater PRL concentrations
in blood than animals receiving 10 to 12 h of darkness daily
(Piacsek et al., 1978; Vaticon et al., 1980). In calves,
when daily duration of light is increased gradually (.38 h
per day) from 8 to 24 h, PRL increases in parallel with
duration of light until 24L:0D is reached, after which PRL
decreases within 1 wk to concentrations that are not
different from concentrations in calves exposed to 8L:16D

(Bourne and Tucker, 1975).- Whether this decrease in PRL is

37

due to abrupt loss of darkness (scotoperiod) or duration
below a critical scotoperiod is unknown. In addition, it is
not known whether a period of absolute darkness is required
to obtain the characteristic PRL response to 8 or 16 h of
light per day. Therefore, experiments were conducted to
ouantify PRL concentrations in bull calves after switching
from 8L:16D to either 16L:8D or to continuous light.

Materials and Methods

 

Animals and Housing. Eight Holstein bulls were used in
each of three experiments as described below. Beginning at
approximately 3 d of age bulls were individually penned in
1.1 by 1.8 m stalls within a light-controlled room. Calves
received 8L:160 for 6 to 10 wk from cool-white fluorescent
lamps (General Electric F40CW/RS/WM). All lamps came on at
0700 h and switched off at 1500 h. An 8L:160 photoperiod
was used to establish low baseline concentrations of PRL in
serum of calves (Leining et al., 1979). Following this
pretreatment conditioning, calves were assigned by weight to
one of two light and temperature controlled chambers (four
calves per chamber). Calves were unrestrained within each
chamber and received a grain concentrate ration (BIR Milking
Chow, Ralston Purina Co., St. Louis, MO), alfalfa hay,
mineral supplement block and water ad libitum. Ambient
temperature in each chamber was maintained at 21 i 2 C.
After 2 wk additional pretreatment exposure to 8L:16D in the
light and temperature controlled chambers, calves were

fitted with an indwelling iugular polyvinyl cannula (Ico-

38

Rally Corp. SLV 105 18 clr) as reported (Leining et al.,
1979). On the following day (dav 0 of experiment) at 0700 h
animals were restrained loosely with halters, and blood was
sampled (10 ml) and discarded at lS-min intervals for l h to
acclimate the animals to the sampling procedure. Starting
at 0800 h, blood was collected at 30-min intervals for 26
(Exp. 1) or 6 h (Exp. 2). Between collection of samples
cannulas were filled with 3.5% sodium citrate to maintain
cannula patency.

Exp. 1. The objective of this experiment was to
compare concentrations of PRL in serum of calves abruptly
switched after the pretreatment period of 8L:16D to 16L:8D
or 24L:00 . At 6 wk of age, mean body weights of four bulls
in each chamber averaged 76.6 and 77.1 i 7.6 kg (means :
S.E.D.). Median light intensities at eye level of standing
calves were 364 and 395 1x.

The day after collection of initial blood samples,
photoperiod was switched from 8L:160 to 16L:8D (0300-1900 h)
in one chamber and to 24L:OD in the other for 9 wk. Calves
were bled three additional times at 3-wk intervals as
described.

Exp. 2. The design of Exp. 1 precluded direct
comparisons between calves of the same age maintained on
8L:16D and calves abruptly switched to continuous light.
Thus, Exp. 2 was conducted to examine the PRL response in
calves abruptly switched from 8L:16D to 24L:0D vs calves

continuously maintained under 8L:16D.

39

Bulls were preconditioned to 8L:16D as previously
described. Mean body weight of four bulls in each chamber
at initiation of the experiment was 111.5 and 111.7 : 8.6
kg. Following preconditioning under 8L:16D, photoperiod was
maintained at 8L:16D in one chamber but switched to 24L:0D
in the other for 8 wk. Median light intensities were 460
and 471 1x. Bulls were bled for 6 h at 30-min intervals on
wk 0, 4 and 8.

Blood Handling and Assay. Blood samples were allowed

 

to clot for 6 to 8 h at room temperature, stored for 24 h at
5 C, then centrifuged at approximately 1000 x g for 20 min.
Serum was decanted and frozen at -20 C until assayed for PRL
as previously described (Koprowski and Tucker, 1973).

Statistical Analysis. For each experiment data were

 

analyzed using split-plot analysis of variance (Gill, 1978b)
to test for differences in concentrations of serum PRL
between and within bleeding periods. For a given treatment
within a 26 or 6 h bleeding period PRL did not differ over
time; therefore, a mean PRL value was calculated for each
animal during each 26 (Exp. 1) or 6 (Exn. 2) h bleeding
period. Within each photoperiod treatment, contrasts
between bleeds were made using Bonferroni-t tests (Gill,
1978b). For the purpose of treatment and bleed comparisons,
variances were expressed as standard errors of differences
(Gill, 1981). See appendix for a listing of sources of
variation and specific contrasts.

Results

40

Exp. 1. After 8 wk pretreatment exposure to 8 h of
light per day PRL averaged 18.6 and 18.5 ng/ml of serum in
the two pretreatment groups (P > .05; figure 3). After
subiecting bulls to 24L:00 or 16L:8D for 3 wk, PRL was
unchanged in bulls receiving 24L:OD but was increased 3.6-
fold in bulls exposed to 16L:8D (P < .01). After 6 wk,
concentrations of PRL in serum increased 2.4- to 3.5-fold in
bulls subiected to 24L:00 or 16L:80 relative to previous
exposure of 8L:16D. Overall, concentrations of PRL in
calves receiving 24L:OD did not differ from concentrations
in calves exposed to 16L:8D over the 9-wk duration of the
experiment (P > .05).

Exp. 2. After 10 wk pretreatment exposure to 8L:16D,
PRL averaged 22.0 and 17.0 ng/ml of serum in the two groups
of four animals (P > .05; figure 4). Four wk after abruptly
switching to 24L:0D, PRL was unchanged and did not differ
from concentrations in calves maintained under 8L:16D.
However, 8 wk after the switch, PRL had increased 1.9-fold
in bulls exposed to 24L:OD compared with bulls maintained
under 8L:16D (P < .05).

Discussion

 

The 3.6—fold increase in concentrations of PRL in serum of
bull calves (Figure 1) following an abrupt shift from 8L:16D
to 16L:8D observed at wk 3 in Exp. 1 confirmed previous
observations using similar protocols (Leining et al.. 1979:
Stanisiewski et al., 1984). Furthermore, it was previously

shown that after a lag of 2 to 3 wk, concentrations of PRL

41

Figure 3.

Prolactin in serum from prepubertal bulls
switched from 8 h of light per day (at wk

0) to continuous (24 L) or 16 h of light per
day. Each observation represents the mean
of four bulls where blood samples were
collected for 26 h through a jugular

‘cannula. Standard error of the difference

between treatments was 27.9 ng/ml of serum
and for wk was 10.6 ng/ml of serum.

42

    

.——-. 24L=OO
.__—-. I6L=80

 

 

 

70r-

I 4 1 1 4
O O O O O
I!) q- IO N '—

(WleBS 4° "MN/9N) NIlOV'lOHd

43

l7

l4

WEEKS

Figure 4.

Prolactin in serum from prepubertal bulls
switched from 8 h of light per day (at wk 0)
to continuous (24 L) or maintained on 8L2l6p.
Each observation represents the mean of four
bulls where blood samples were collected for

6 h through a iugular cannula. Standard error
of the difference between treatments was 5.5
ng/ml of serum and for wk was 4.6 ng/ml of
serum.

44

 

 

mxwmts

 

 

m_ N.
_ _
10.
O. IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII ION
. nnnnnnn 4....
10m
10¢
cm. “.5 9......
oouqvmolo .on
Low

(wnaas 40 'IIN/ON) NIlOV'lOHcI

4S

in serum of bull calves gradually increased in parallel with
gradual increases in duration of light between 8L and 24L
only to be followed by a decrease as 24 L continued (Leining
et al., 1979). In the present series of experiments,
concentrations of PRL in serum increased when photoperiod
was abruptly switched from 8L:160 to continuous light.
However, in Exp. 1, calves abruptly switched from 8L:16D to
16L:80 achieved maximum concentrations of PRL after 3 wk,
whereas switching to continuous light required 6 wk before a
peak in serum PRL was seen. A similar observation was made
in Exp. 2 in which a total of 8 wk were required after the
switch from 8 to 24L before a significant increase in PRL
was detected. Absolute increments of PRL in response to 24
L were not the same in Exp. l and 2: however, responses of
different groups of calves to identical photoperiods are
rarely similar in terms of magnitude of PRL change
(Stanisiewski et al., 1984). The data are consistent with
the hypothesis that daily dawn-dusk (light-dark) transitions
provide an external cue used to mediate seasonal changes in
the physiology of mammals (Pelletier et a1., 1985). The
lack of a daily dawn-dusk transition in bulls abruptly
switched to continuous light may explain the slower
responsiveness in terms of increased serum concentrations of
PRL. The mechanism whereby continuous light causes an
eventual increase in concentrations of PRL in bulls is not
known, but I speculate that it may be associated with a

circannual rhythm analogous to that observed in circadian

46

rhythms (Simpson and Follett, 1982). Rats receiving 24L:OD
demonstrate a rhythm of PRL secretion with a periodicity of
6 to 8 h (Bethea and Neill, 1979) but no such rhythm was
detected in my calves, as differences at 30 min intervals
were small. In the absence of photoperiodic and seasonal
cues, Petitclerc et al. (1983b) observed an endogenous
circannual rhythm of PRL secretion in cattle. However, the
duration of present experiments were not sufficient to
detect a complete cycle of hormone activity. Therefore, I
do not knowwhether the animals were exhibiting a circannual
rhythm.

When compared with 12 to 14 h daily dark exposure, male
rats receiving 24L:OD for 7 d have lower PRL concentrations
in blood (Relkin, 1972), whereas male and female rats
receiving 24L:OD for 41 to 60 d have higher PRL in blood
(Piacsek et al., 1978; Vaticon et al., 1980). Likewise, in
my calves, continuous light must be applied for 6 to 8 wk
before PRL increases. Furthermore, PRL enhances
reproductive development in male rats (Moger et al., 1972).
In fact, male rats exposed to 24L:OD from birth have larger
testis by d 55 than rats exposed to periods of daily
darkness (Hoffmann, 1973). But whether the ability of
photoperiod to hasten sexual maturation in cattle
(Petitclerc et al., 1983a: Hansen et al., 1983) is mediated
in part through PRL remains to be determined.

The tendency (P < .1) for concentrations of PRL to

decline in Exp. 1 despite maintenance of 16L:8D or

47

continuous light is consistent with the hypothesis that
animals may become refractory to a given repetitive
photoperiodic signal (Sharp and Moss, 1977: Follett et a1.,
1975) Decline in concentrations of serum PRL in response to
continuous lighting has been reported previously (Leining et
al., 1979).

In summary, calves receiving constant light for more
than 3 wk have greater concentrations of PRL in serum than
calves exposed to 8 h of daily light. However, maximal PRL
concentrations can be achieved sooner if a 16L:8D
photoperiod is provided. I found that continuous light
photoperiods stimulate PRL secretion in bull calves relative
to 8L:16D, but the time course to achieve maximum PRL
concentrations in serum is slower with continuous light vs

16L:8D.

48

Chapter 2

Effect of Photoperiod and Castration on Prolactin,

Testosterone and Luteinizing Hormone Concentrations

in Male Calves.

49

Introduction

 

Switching photoperiod from 8L:16D to 16L:8D increases
serum PRL concentrations in prepubertal bulls 2- to 8-fold
(Bourne and Tucker, 1975: Stanisiewski et al., 1984). In
previous experiments with cattle comparing 8L:16D with
16L:80, maximum duration of exposure to a single photoperiod
was 6 wk (Stanisiewski et al., 1984). The effect of
photoperiods maintained longer than 6 wk on PRL is unknown.
In addition, rams exposed to decreasing daily light
decreased PRL but increased LH and testosterone
concentrations in plasma (Lincoln et al., 1977: Sanford et
al., 1978). Previous researchers failed to detect a
photoperiod effect on LH concentrations in cattle (Bourne
and Tucker, 1975: Rzepkowski et al., 1982). However,
progesterone concentrations in serum of young heifers
exposed to long days increase at an earlier age relative to
heifers receiving short-day photoperiods (Petitclerc et al.,
1983a), which suggests that long-day photoperiods may hasten
onset of puberty in heifers.

My objective was to examine long term effects of long-
vs short-day photoperiods on PRL, LH and testosterone
secretion in bull calves. A second objective was to
determine if PRL response to photoperiod in prepubertal
bulls was dependent upon the testes.

Materials and Methods

 

Pre-treatment. In each of two experiments (1 and 2)

eight Holstein bulls (approximately 3 d of age) were placed

50

into a light-controlled room as previously described
(Stanisiewski et a1., 1984). Fluorescent lights were
programmed on at 0700 h and off at 1500 h (8L:16D). After
weaning at approximately 6 wk of age, calves were moved to
one of two light-and temperature-controlled chambers
described previously (Stanisiewski et al., 1984), where
ambient temperatures were maintained at 21 :p2C during Exp.
1 and at 15 : 1C during Exp. 2. Photoperiod was then
maintained at 8L:16D for 2 additional wk. Thus,
pretreatment photoperiod lasted a total of 8 wk. Calves
were fed a concentrate mixture plus alfalfa hay and water ad
libitum.

In Exp. 2, testes were removed surgically from four
calves at 2 wk of age. Four additional calves were
maintained as gonadally intact controls.

Treatments. In Exp. 1, four calves were maintained on

 

8L:16D, whereas four calves were switched to 16L:80 (lights
on from 0300 to 1900 h) for 12 additional wk. In Exp. 2,
photoperiod was switched from 8L:16D to 16L:8D on all calves
(four castrates, four gonadally intact controls) for 6 wk.
Median light intensities measured 1 m above the floor were
equalized between chambers within each experiment (S46 1x,
Exp. 1: 517 1x, Exp. 2).

Blood Sampling. Two d prior to start of treatments,
calves were fitted with an indwelling jugular cannula (SLV
105 18 clr)3 and bled the following day. On day of

sampling, animals were loosely restrained by halter at 0700

51

h and blood samples were discarded at 15-min intervals for l
h to accustom animals to the sampling procedure. Beginning
at 0800 h, 8 to 10 ml of blood was collected for 6 h at 30-
min intervals. All calves had been exposed to a total of 8
wk of 8L:16D at the time of this first blood collection. In
Exp. 1, blood was sampled again.at wk 14 and 20 of age: in
Exp. 2, blood was sampled again at wk 14.

Sodium citrate (3.5%) was used to prevent coagulation
of blood in cannulas between collection of samples. Blood
samples were allowed to clot for 6 to 8 h at room
temperature, stored for 24 h at 5C, then centrifuged at
approximately 1000 x g for 20 min. Serum was decanted and
frozen at -20C until assayed.

Hormone Assays. Previously described radioimmunoassays
were used to quantify PRL (Koprowski and Tucker, 1971), LH
(Convey et al., 1976) and testosterone (Haynes et a1., 1977)
in serum.

Statistical Analysis. Data from each experiment were
analyzed using split-plot analysis of variance (Gill, 1978b)
to test for differences in concentrations of serum PRL, LH,
and testosterone between and within bleeding periods. Main
effects were photoperiod treatment and day of bleeding.
Hormone values were averaged over each 6-h sampling period
to obtain a representative mean. Bonferroni t test (Gill,
1978a) was used to test for differences between means of
treatments and bleeding days. Frequency of LH and

testosterone pulses per 6-h were determined using a pulse

52

analysis program (PULSAR; Merriam and Wachter, 1982). See
appendix for a listing of sources of variation and specific
contrasts.

Results

Exp. 1. After 8 wk pretreatment with 8L:16D, PRL
averaged 17.0 and 19.6 (i 4.2, pooled SE) nq/ml (P > .05) of
serum for both groups of four animals (figure 5). PRL in
serum was greater (P < .02) in calves exposed to 6 wk of
16L:8D than in calves maintained under 8L:16D [93.8 vs 36.9
(i 16.1) ng/ml: figure 51. However, after 12 wk exposure to
16L:8D (20 wk of age), PRL in serum declined and was not
different (P > .05) from PRL in bulls exposed to 8L:16D
[34.7 and 17.2 (i 6.4) ng/ml: figure 5]. In calves
maintained under short days, PRL did not change (P > .05)
throughout the experiment.

Concentrations of LH were not different (P > .05)
between photoperiod treatments nor between bleeding periods
(figure 6). Pooled among treatments, LH averaged .80 i .16,
1.06 i .13 and .73 i .20 ng/ml of serum for the eight calves
at 8, 14 and 20 wk of age (figure 6). Frequency of LH
pulses per 6-h were unaffected (P > .25) by photoperiod
treatment and did not change with age, averaging 2.9, 2.9
and 2.1.

Testosterone in each group of four bulls after
pretreatment with 8L:16D for 8 wk averaged .39 and .47 (i
.03) ng/ml of serum (P > .05, figure 7). Six wk later (age

14 wk), testosterone in serum was greater (P < .05) in

53

Figure 5.

Prolactin in serum from bull calves exposed‘
to 8 h of light per day from 3 d to 8 wk of
age and subsequently switched to 16 h of
light per d or maintained on 8L:16D. Each
observation represents the mean of four bulls
where blood samples were collected at 30-min
intervals for 6 h through a jugular cannula.
Standard error of the difference for treat-
ments was 23.9 ng/ml of serum and for wk of
age was 9.8 ng/ml of serum.

54

 

 

IOOF-

20

. I. nun-IS:
CDC)
9°22
4;:
52(1)
\
I -CD
I l l L
O O O O O
m (D Q’ N

(wnaas 1° IUJ/5U) NIlOV'IOHd

55

AGE (WEEKS)

Figure 6.

Luteinizing hormone in serum from bull calves
exposed to 8 h of light per day from 3 d to 8
wk of age and subsequently switched to 16 h
of light per d or maintained on 8L:16D. Each
observation represents the mean of four bulls
where blood samples were collected at 30-min
intervals for 6 h through a jugular cannula.
Standard error of the difference for
treatments was .23 ng/ml of serum and for

wk of age was .19 ng/ml of serum.

56

Amxwwgv mud

ON v. m

_ _ . _

../.\.

023m nTIO
09.5. I

 

ON

(wnaas Io IUJ/bu) H'l

57

Figure 7.

Testosterone in serum from bull calves exposed
to 8 h of light per day from 3 d to 8~wk of
age and subsequently switched to 16 h of light
per d or maintained on 8L:160. Each
observation represents the mean of four bulls
where blood samples were collected at 30—min
intervals for 6 h through a jugular cannula.
Standard error of the difference for treat-
ments was .23 ng/ml of serum and for wk of

age was .29 ng/ml of serum.

58

Amxmmzs mo<

ON v. m .
_ _ . _ o

 

020.5 DID
calm. IIII

 

(wnuas 40 "wow BNOHBISOISBI

S9

calves receiving 16L:8D (1.92 i .19 ng/ml) than in calves
maintained under 8L:16D (.97 i .19 ng/ml, figure 7). By wk
20 of age, testosterone in serum was numerically greater in
16L:BD exposed bulls but not statistically different (P >
.05) from testosterone in bulls given 8L:16D (2.36 and 1.83
i .21 ng/ml). Frequency of testosterone pulses per 6-h were
unaffected (P > .25) by photoperiod treatment and did not
change (P > .25) with age, averaging 2.0, 2.5 and 2.0 at wk
8, 14 and 20 of age.

Exp. 2. PRL in serum was not different (P > .05)
between castrated (12.5 ng/ml) and intact (12.1 ng/ml)
calves pretreated with 8L:16D through 8 wk of age (figure
8). Subsequently, PRL concentrations in serum after 6 wk of
16L:8D increased (P > .01) similarly in castrated and intact
calves to (48.0 and 59.8 (i 12.7) ng/ml, (P > .05)
respectively. LH in serum was greater (P < .01) in
castrated (3.0 i .35 ng/ml) than in intact (1.4 i .35 ng/ml)
calves 6 wk after castration. LH in serum was unchanged (P
< .19) after 6 wk of 16L:8D in castrated (3.6 i_.27 ng/ml)
and intact (1.7 i .27 ng/ml) calves.

Discussion

 

I previously showed that abruptly switching
photoperiods of calves from short-to long-days linearly
increased serum PRL concentrations with the maximum
occurring within 6 wk after the switch (Stanisiewski et al.,
1984). Data in the present study confirm this response. In

addition, I have now demonstrated that long-day induced

60

Figure 8. Prolactin in serum from calves exposed to 8 h
of light per day from 3 d to 8 wk of age and
subsequently switched to 16 h of light per
day. Each observation represents the mean of
four bulls where blood samples were collected
at 30-min intervals through a jugular cannula
for 6 h. Castrations were performed at 1.5
to 2 wk of age. Standard error of the
difference for treatments was 14.1 ng/ml of
serum and for wk of age was 11.1 ng/ml of serum.

61

 

 

loo--

.0 15".
E
,_<
QC
<1—
»2
50
TO
.3; -
O-CD
l l l l
O O O O O
Q (0 V N

(wnaas 1° luv/60) NIlOV‘IOHd

62

AGE (WEEKS) .

increases in PRL concentrations (at 6 wk after switch) are
not maintained indefinitely, but decline to concentrations
that are not significantly different from those in calves
maintained under short-days. Thus, elevated PRL
concentrations in bull calves become refractory to a once
stimulatory photoperiod. Photorefractoriness as reflected
by PRL secretion and gonadal function has been documented in
sheep (Almeida and Lincoln, 1984) and hamsters (Bittman,
1978). In seasonal breeding species such as sheep and
hamsters, photorefractoriness serves as an inherent control
mechanism whereby reproduction is limited to specific
seasons (Robinson et a1., 1985). On the other hand, the
reason bull calves become refractory to long-days in terms
of PRL secretion and what function this serves is unclear.
Circulating concentrations of testosterone normally
increase linearly between 8 and 24 wk of age in bulls
(Bedair and Thibier 1979; Amann and Walker, 1983). My
results are in agreement with this pattern. In addition, I
discovered that long-days increase the rate at which serum
testosterone increases. Therefore, I speculate that puberty
onset may be achieved at an earlier age in bulls exposed to
long-days when compared with bulls exposed to short-day
photoperiods. Duration of photoperiod hastens onset of
puberty in heifers (Petitclerc et al., 1983a), boars (Mahone
et al., 1979), rams (Howles et al., 1980), deer (Budde,
1983) and hamsters (Hoffmann, 1978). Photoperiod control of

sexual development at puberty plays an important role in

63

seasonal breeders in which reproductive activity is limited
to certain times of the year, but the function of this
maturational response in nonseasonal breeders such as cattle
is not known.

Long-day photoperiods stimulate growth rates and
protein deposition in cattle (Tucker et al., 1984) and sheep
(Forbes et al., 1979). In cattle, these effects require the
gonad (Tucker et al., 1984). Since, in the present study,
long days induced an early increase in serum concentrations
of testosterone and testosterone stimulates growth in
ruminants (Hafs et al., 1971: Schanbacher et al., 1980) I
suggest that testosterone may at least be a partial mediator
of photoperiod effects on growth in bulls. Because of few
animal numbers in the present study, photoperiod treatment
effects on body growth were not measured.

As reported previously in cattle (Bourne and Tucker,
1975: Rzepkowski et a1., 1982), LH concentrations in
castrated and intact bulls were unaffected by photoperiod.
In adult bulls, LH controls testosterone secretion (Hafs and
McCarthy, 1980); however, in bulls younger than 20 wk of
age, serum LH is not closely coupled to testosterone
secretion (Karg et a1., 1976: Lacroix and Pelletier, 1979).
Therefore, it is not surprising that concentrations of LH
were not associated with long-day stimulation of
testosterone secretion in my experiment. In fact, frequency
of LH and testosterone pulses did not change throughout Exp.

1, and pulses between the two hormones were usually

64

asynchrouous. Furthermore, PRL is capable of increasing LH
receptors in rat (Morris and Saxena, 1980) and hamster (Bex
et a1., 1978) testes, but similar effects are not seen in
rams (Barenton, 1981). In rats, hyperprolactinemia
transiently increases circulating testosterone (Waeber et
a1., 1983) while LH concentrations are unchanged. Perhaps
long-day induced PRL concentrations increase LH receptors in
prepubertal calf testes, and thus may potentiate the effects
of LH on testosterone secretion.

In the present study I showed that long-days increased
concentrations of PRL in serum 4- to 5-fold compared with
previous short-day exposure, and this response occurred
independent of the testes. The observed increase in PRL was
well within the 2- to 8-fold range previously reported
(Bourne and Tucker, 1975; Stanisiewski et a1., 1984).
However, I do not know whether long-day induced increases in
PRL are causally associated with increased secretion of
testosterone. To test this possibility in a preliminary
study, four bull calves received a long-day photoperiod
while four similar calves received a short-day photoperiod.
Two long-day exposed bulls were given 2-Br- ergocryptine
(CB-154) to reduce PRL secretion. After 4 wk of treatment,
PRL averaged 2.8, 43.1 and 23.0 ng/ml of serum in long-day
plus CB-154, long-day and short-day treated animals,
respectively. Concurrently, testosterone in serum averaged
.51, .95 and .85 ng/ml for the same respective treatments.

Testosterone was lowest in calves with lowest PRL

65

concentrations. However, animal numbers were too few and
animals may have been too young to detect significant
differences. The trend supports the hypothesis that
photoperiod-induced testosterone increases were associated
with increased secretion of PRL, however, more animals are
needed to substantiate the trend.

In summary, long-days stimulated PRL secretion relative
to short-days in bull calves, but the stimulation was not
maintained indefinitely. This response occurred independent
of the gonad. I speculate that onset of puberty may also be
hastened in bulls exposed to 16L:8D because testosterone in
serum of these bulls was elevated at an earlier age than in
bulls given 8L:16D. This early increase in testosterone was

associated with PRL but not LH.

66

Chapter 3

Prolactin and Catecholamine Concentrations in Blood and
Pituitaries of Prepubertal Bulls Exposed to 16 or 8 Hours of

Light per Day

67

Introduction

 

Concentrations of PRL in serum of bull calves are
increased about 5-fold when photoperiod is switched from
8L:16D to 16L:8D compared with calves maintained on 8L:16D
(Stanisiewski et al., 1984). The neuroendocrine mechanism
controlling photoperiod-induced PRL secretion is unknown.
However, evidence indicates that PRL release from the
anterior pituitary gland is tonically inhibited by dopamine
(DA) (Leong et al., 1983), and possibly other catecholamines
(Shaar and Clemens, 1974). Both DA receptor binding and
hypothalamic DA concentration vary with season in rats
(Naber et al., 1981) and humans (Karson et a1., 1984).
Thyrotropin-releasing hormone (TRH) stimulates PRL release
in vitro and in vivo (Beck et al., 1979, Smith and Convey,
1975) and is considered a principal physiological regulator
of PRL. Furthermore, TRH-induced PRL release is greater in
sheep (Howland et a1., 1983) and calves (Leining et a1.,
1979) exposed to long days compared with short days.
Therefore, our objective was to determine effects of 16L:8D
and 8L:16D on concentrations of DA, norepinephrine (NE) and
epinephrine (EPI) in the pituitary and on release of PRL
from pituitaries of prepubertal bulls.

Materials and Methods

 

Animals and Housing. Beginning at approximately 3 d of
age, eight bull calves were penned individually in a light-
controlled (8L:16D) room. This pretreatment period was used

to establish low baseline PRL concentrations in serum

68

(Stanisiewski et a1., 1984). Specific housing conditions
and diets were previously described (Leining et al., 1979).
At about 7 wk of age, calves were assigned by weight
(average 71.4 i 2.2 kg) in to one Of two light and
temperature controlled chambers (four calves per chamber).
Two wk later (wk 0 of experiment) calves were fitted with an
indwelling jugular cannula as described (Leining et al.,
1979) and bled (lO-ml per sample) the following d from 0700
h to 0800 h (15-min interval presampling) and thereafter at
30-min intervals for 6 h. Ambient temperatures were 21.3 i
.8 C. Four calves then received 16L:8D for 6 additional wk
while the other four calves were maintained on 8L:16D. At
the completion of the experiment (wk 6), calves were bled
again as described and slaughtered the following d at a
local abattoir, where pituitaries were removed from each
calf within 10 min of death.

Blood Handling and Assay. At the time of collection of
each blood sample , approximately 2 ml of blood was
centrifuged at 1000 x g for 10 min and 500 ul of resulting
plasma was added to 50 ul .2N perchloric acid and frozen
immediately on dry ice until assayed (radioenzymatic) for
total DA, NE and EPI (Peuler and Johnson, 1977: Umezu and
Moore, 1978). Remaining portions of samples were allowed to
clot for 6 to 8 h at room temperature, stored for 24 h at 5
C, then centrifuged at 1000 x g for 20 min. Serum was
decanted and frozen at -20 C until assayed for PRL as

previously described (Koprowski and Tucker, 1973).

69

Tissue Handling and Incubation. Pituitaries were

 

removed from calves and placed on ice. Approximately 20 min
later, a medial slice of posterior and anterior pituitaries

was frozen (—70 C) until assayed for DA, EPI and NE content

as described above. Remaining anterior pituitary glands

2 explants and about 20

from each calf were sliced into 1 mm
mg (weights recorded) of tissue was transferred to 3 ml
Medium 199. Tissues were washed at lS-min intervals for 1 h
then incubated for 1.5 h in a Metabolic Shaking Incubator
(Precision Scientific, Chicago, IL) at 37 C under oxygen.
Media were harvested (pre-treatment) for quantification of
basal PRL concentrations released. In a 2 by 3 factorial
experimental design, tissue from calves exposed to each
photoperiod was incubated for an additional 2 h with TRH (12
ng/ml of media), DA (60 ng/ml of media) or vehicle (medium
199). Concentrations of TRH and DA are within reported
ranges to satisfactorily stimulate or inhibit PRL release
from pituitaries in vitro (Smith and Convey, 1975; Fagin and
Neill, 1981). Treated media were decanted (post—treatment)

and stored (—20 C) until assayed for PRL.

Statistical Analysis. Split-plot analysis of variance

 

(Gill, 1978b) was used to compare hormone concentrations in
blood, as well as PRL concentrations in media. PRL release
into media was expressed as pre-to post-treatment
difference. Catecholamine contents in pituitaries were
analyzed by Chi Square analysis (Gill, 1978a). See appendix

for a listing of sources of variation and specific

contrasts o

70

Results

Prolactin in Blood. At wk 0 of the experiment, PRL

 

averaged 43 ng/ml of serum over 6 h in each group of calves
receiving 8L:16D. PRL was unchanged (P > .1) 6 wk later in
calves maintained under 8L:16D (32 ng/ml of serum) but was
increased (P < .01) after 16L:8D treatment (75 ng/ml of
serum).

Catecholamines in Blood. Concentrations of

 

catecholamines in blood were quantified at the end of 6 wk
treatment with 8L:16D or 16L:8D. DA declined (P < .01)
throughout the 6 h sampling period in all calves averaging
.33 and .36 ng/ml of plasma (P > .25) in 8L:16D and 16L:8D
exposed calves (figure 9). NE averaged .41 and .44 ng/ml
over the 6 h and were not significantly different (P > .25).
and EPI averaged .26 and.25 ng/ml (P >.25) in 8L:16D- and
16L:8D-exposed calves, respectively, (figure 10, ll).

Catecholamines in Pituitaries. Anterior pituitary DA
tended to be greater (P < . 16) in calves exposed to 8L:16D
(.35 ng/mg of protein) than in 16L:8D-exposed calves (.21
ng/mg of protein: table 1). NE was not detectable and EPI
in anterior pituitaries was not different (P > .25) between
photoperiod treatments (table 1). Posterior pituitary
concentrations of DA, NE and EPI concentrations did not
differ (P > .25) between photoperiod treatments.

In Vitro PRL Release. Pre-treatment PRL concentrations
averaged 440 and 326 ng/ml medium'mg-1 pituitary (P > .25)

in 8L:16D- and 16L:8D-exposed calves, respectivelV. (table

71

Figure 9.

Dopamine in plasma from prepubertal bulls
receiving 8 or 16 h of light per day.

Each observation represents the mean of four
bulls where blood samples were collected for
6 h through a jugular cannula.

72

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73

HOURS

Figure 10.

Norepinephrine in plasma from prepubertal
bulls receiving 8 or 16 h of light per day.
Each observation represents the mean of four
bulls where blood samples were collected for
6 h through a jugular cannula.

74

L
m

o'

BLZIGD
--- l6L3 80

1 I

vs 0. to. <r. m. N.
000000

(DLUSOId Nil/5”) 3Nl8Hd3NId380N

 

75

 

HOURS

Figure 11.

Epinephrine in plasma from prepubertal bulls
receiving 8 or 16 h of light per day. Each
observation represents the mean of four bulls
where blood samples were collected for 6 h

through a jugular cannula.

76

l l 1 l l

       

 

 

 

L I
“I E Q Q E 0 N
o o o o o o o'

(DwSOId Iw/DU) BNIHHdBNIdB

77

HOURS

Table 1. PITUITARY CATECHOLAMINE CONCENTRATIONSa

 

ITEM 16L:BD 8L:16D P

Anterior pituitary DA .21 .35 (.16
NB - -

EPI .06 .05 NS

Posterior pituitary DA 2.58 2.52 NS

NE .26 .14 NS

EPI .13 .16 NS

 

a ng/mg protein.

78

Table 2. PRL RELEASE FROM ANTERIOR PITUITARY EXPLANTSa

 

 

 

16L:80 8L:16D
Basal release 326 440
Treatment challengeb
Control 0C OC
TRH +141d +90d
DA -sse -105e

 

a ng/ml medium‘mgm1 pituitary.

b Data expressed as change from control.

c,d,e. Values with different superscripts differ (P<.01)-

79

2). PRL concentrations were unchanged (P > .25) in media
from pituitaries challenged with vehicle, but relative to

controls TRH increased (P < .01) PRL release 90 and 141

1

ng/ml medium.mg- pituitary (P > .2) in explants from 8L:16D

and 16L:8D exposed calves, respectively: (table 2).

Conversely, addition of DA to media decreased (P < .01) PRL

release compared with controls I-lOS ng/ml.mg-1, 8L:16D

and -55 ng/ml.mg-1, 16L:8D (P > .2)].

Discussion

 

In agreement with previous studies (Leining et al.,
1979: Stanisiewski et a1., 1984), PRL concentrations in
serum were increased by 16L:8D exposure relative to 8L:16D.
Photoperiod is a major contributor to seasonal variability
of PRL concentrations in cattle (Koprowski and Tucker, 1973;
Petitclerc et al., 1983b). Consequently, in vitro release
and pituitary gland content of PRL correspond to seasonal
changes in circulating concentrations of the hormone in
cattle (Dular and LaBella, 1977). I now have evidence that
DA may be involved in photoperiod-modulated PRL release
since DA concentrations were greatest in anterior pituitary
glands of calves with lowest circulating PRL concentrations.

Van Maanen and Smelik (1968) first proposed the concept
that the monaminergic tubero-infundibular neural system was
responsible for direct control of PRL secretion.

Measurement of DA in hypophyseal stalk plasma in quantities
sufficient to inhibit PRL release substantiated this
hypothesis (De Greef and Neill, 1979). Furthermore,

Gudelsky et a1. (1980) suggested
80

that 75 to 85% of DA in the anterior pituitary gland is
associated with secretory granules (lactotrophs) containing
PRL. I propose that the difference in pituitary gland DA
concentrations is a chronic response to several weeks of
photoperiod treatment. Further experiments are needed to
determine whether changes in anterior pituitary DA
concentrations are closely coupled with change in serum
concentrations of PRL in response to photoperiod.

Others have shown that TRH-induced PRL release is
greater in bulls (Leining et a1. 1979) and ewes (Howland et
a1., 1983) exposed to long as compared with short days.
Furthermore, I found that in vitro TRH-induced PRL release
was numerically greater (57%) from pituitary glands
previously exposed to 16L:8D compared with 8L:16D exposure
although this preliminary result needs to be confirmed.
However, if TRH increases PRL secretion in vitro more in
16L:8D— than in 8L:16D-exposed calves, then perhaps a
chronic photoperiod-induced change in the lactotrophs has
occurred. It is unknown whether this change is associated
with change in number of lactotrophs or sensitivity of
lactotrophs to PRL releasing or inhibiting factors. Since
TRH and DA probably mediate PRL secretion through opposing
effects on intracellular signals within lactotrophs (Ray and
Wallis, 1984), higher DA concentrations in 8L:16D exposed
pituitary glands may partially explain photoperiodic

differences in TRH-induced PRL release.

81

Chapter 4

Effect of Pinealectomy on Prolactin, Testosterone and

Luteinizing Hormone Concentrations in Plasma of Bull

Calves Exposed to 8 or 16 Hours of Light per Day

83

Introduction

 

PRL concentrations in serum are greater in calves
exposed to 16L:8D than in calves exposed to 8L:16D (Bourne
and Tucker, 1975: Stanisiewski et al., 1984). .In addition,
testosterone concentrations in peripubertal bulls increase
at an earlier age with 16L:8D exposure than with 8L:16D
(Stanisiewski et al., 1987b). Concentrations of LH in serum
of cattle are unaffected by photoperiod (Bourne and Tucker,
1975: Rzepkowski et al., 1982).

The mechanism whereby photoperiod affects hormone
concentrations in cattle is not understood, but in sheep,
removal of the pineal gland abolishes normal photoperiod-
induced changes in PRL (Brown and Forbes, 1980: Brinklow and
Forbes, 1984) and testosterone (Barrell and Lapwood, 1979).
Pinealectomy of young steers reduces 16L:8D-induced
increments in PRL concentrations in serum, whereas blinding
completely abolishes the response (Petitclerc et al.,
1983b). The pathway for signal transmission between the
pineal gland and anterior pituitary gland is less clear:
however, others have suggested that the indolamine,
melatonin, likely acts as a hormonal transducer (Goldman and
Darrow, 1983). Melatonin is synthesized primarily in the
pineal gland, and serum concentrations are greatest during
darkness (Goldman and Darrow, 1983).

My objective was to examine the effect of pinealectomy
on PRL, testosterone and LH in peripubertal bulls exposed to

8 or 16 h of light per day.

84

Materials and Methods

Animals and Housing. The following housing conditions
and treatments were followed using two replicates of eight
Holstein bulls. Eight bulls approximately 3 d of age were
placed into a light-controlled room as previously described
(Stanisiewski, et al., 1984). All calves received 8L:16D
daily (lights on 0700 h) through weaning at approximately 6
wk of age. Within replicate, four calves were
pinealectomized (PX) on average at 5.6 wk of age while four
additional calves were sham pinealectomized (SPX) using
surgical procedures previously reported (Braun et al.,
1977). Excised tissue was examined histologically to
confirm removal of pineal tissue. Surgeries were performed
at the Michigan State University Veterinary Clinical Center
where calves remained under constant light for approximately
1.5 wk while recovering from surgery. Calves were then
returned to 8L:16D light exposure. At approximately 18 wk
of age, calves were assigned to one of two light-and
temperature-controlled chambers as described (Stanisiewski
et a1., 1984) and fed a commercial calf grain diet plus
alfalfa hay ad libitum. Body weights were 116.1 : 6.4 kg
(mean : S.E.) at the time of chamber assignment. Within
replicate there were two PX and two SPX calves per chamber
and photoperiod remained at 8L:16D for 2 additional wk.
Ambient temperatures were 22.1 :_1.8C for the first
replicate of eight animals and at 20.9 i_.6C for the second

replicate. Median light intensities in chambers averaged
411 1x 1 m above the floor.

85

Photoperiod Treatment. Within replicate beginning at

 

approximately 20 wk of age (wk 0 of experiment), two PX and
two SPX calves received 16L:8D daily for 12 wk while at the
same time two PX and two SPX calves were maintained on
8L:16D.

Blood Sampling. Two d prior to initiation of

 

photoperiod treatments at wk 0, calves were fitted with an
indwelling jugular catheter connected to a 3 m cannula and
the cannula was passed into an adjoining room. Thus, blood
could be sampled remotely without disruption of calves'
ambient photoperiod/scotoperiod. On the day prior to
initiation of photoperiod treatment at wk 0, calves were
pre-sampled (blood was discarded) beginning at 1300 h for l
h and thereafter bled for 26 h at 30-min intervals. All
calves had been exposed to approximately 14 wk of 8L:16D at
the time of this first collection of blood. Calves were
recannulated blood was sampled again at wk 4, 8 and 12.
Ambient temperatures were recorded at the time of each
sample collection.

Sodium citrate (3.5%) was'used to prevent coagulation
of blood in cannulas between sample collections. Blood
plasma was collected using .25 m1 of a 30% sodium citrate
solution per 10 ml of blood and stored at 5C. Samples were
centrifuged at approximately 1000 x g for 20 min and plasma
was frozen at -20C until assayed.

Hormone Assays. Previously described radioimmunoassays

 

were used to quantify PRL (Koprowski and Tucker, 1971),

86

testosterone (Haynes et al., 1977) and LH (Convey et al.,
1976) concentrations in plasma. Direct assay of plasma
concentrations of melatonin were performed using the
procedure and antibody of Fraser et a1. (1983). To validate
this assay in our laboratory, melatonin was quantitatively
recovered (100 pg: 91.0 i 4.7%, 500 pg: 94.7 i 3.8%, 1000
pg: 90.6 i 7.3%: mean : S.E., n = 6). In addition, assay
parallelism was tested using three serum samples at
dilutions compatable with the usable range of the standard
curve (table 3).

Statistical analysis. Main effects (replicates,
surgical treatment, photoperiod treatment and week on
experiment) on hormone concentrations were analyzed using
split-plot analysis of variance (Gill, 1978b). Subsequent
analyses were performed using Bonferroni t test (Gill, 1986)
to examine differences between treatments and week on
experiment. To account for the range of ages among calves
and ambient temperature differences between replicates (see
p. 83), age and ambient temperature were used as covariates
to adjust testosterone and PRL concentrations, respectively.
See appendix for a listing of sources of variation and
specific contrasts.

Results

At wk 12 of experiment, melatonin in plasma of PX
calves receiving 16L:8D averaged 18.9 pg/ml with lights on
and 21.9 pg/ml with lights off (P > .25: figure 12). In SPX

calves receiving 16L:8D, melatonin averaged 15.8 pg/ml when

87

Table 3. CONCENTRATIONS OF MELATONIN IN DIFFERENT VOLUMES
OF BOVINE SERUM

 

 

Volume of Calculated
Sample no.a serum (ul) melatonin (pg/ml)
l 100 56
200 49
2 200 84
300 86
400 93
3 100 192
200 248
300 225

 

a . .
There were 51x replicates per sample.

88

lights were on and 92.0 DO/ml when lights were of? (Figure
12). One PX calf receiving 8L:16D had average lightzdark
melatonin values of 6.5 and 5.4 nO/ml of plasma while the
remaining three PX calves in this treatment demonstrated a
dark-induced rise in melatonin From 16.7 during lights on to
60.1 pg/ml oF plasma during the dark period (Figure 13).
Since these three calves were apparently incompletely
pinealectomized, data from these calves were deleted From
calculations. With lights on, melatonin averaged 16.6 pQ/m]
of plasma in SPX calves on 8L:16D and increased (P < .05) to
71.2 pg/m] when lights were off (figure 13).

Concentrations of PRL in plasma differed (P < .05)
between the two replicates of eight animals, but this
difference was eliminated when data were adiusted for
differences in ambient temperatures. Therefore, replicate
was eliminated From the statistical model.

After 14 wk of 8L:16D treatment (wk 0 of the
experiment), PRL averaged 47.1 ng/ml 0? plasma among calves
(Figure 14). Concentration of PRL in plasma was unaffected
(P > .25) by surgical treatment. At wk 4 of experiment, PRL
increased (P < .01) in plasma of PX (79.8 ng/ml) and SPX
(96.3 ng/ml) calves receiving 16L:8D while remaining
unchanged (P > .25) in PX (52.8 ng/ml) and SPX (47.8 ng/ml)
calves maintained on 8L:16D (figure 14). Prolactin
concentrations in plasma remained higher (P < .05) in calves
receiving 16L:8D compared with calves maintained on 8L:16D

through wk 12 of experiment.

89

Figure 12.

Twenty-six h profiles of melatonin in plasma
of bull calves receiving 16L:80 after 12 wk
on experiment. Each observation represents
the mean of four bulls. Samples were col-
lected at l-h intervals through a iugular
cannula. The hatched bar along the x-axis
represents darkness. Standard errors of
means during light and darkness; respect-
ively, were 6.8 and 21.8 pg/ml of plasma
for SPX calves and 6.3 and 6.0 for PX calves.

90

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91

Figure 13.

Twenty-six h profiles of melatonin in plasma
of bull calves receiving 8L:16D after 12 wk
on experiment. Each observation represents
the mean of four (SPX), three (Incomplete PX)
or one (PX) bulls. Samples were collected at
1-h intervals through a jugular cannula.
Hatched bars along the x-axis represent dark-
ness. Standard errors of means during light
and darkness: respectively, were 8.3 and 35.6
pg/ml of plasma for SPX calves, 6.9 and 27.2
pg/ml of plasma For incomplete PX calves and
6.4 and 5.4 DO/ml of plasma for the PX calf.

92

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93

[‘1‘

Figure 14.

Prolactin in plasma from PX and SPX bull
calves exposed to 8 h of light per day (at
wk 0) and subseguently switched to 16 h of
light per day or maintained on 8L:16D. Each
observation represents the mean of Four
bulls except the treatment combination of
8L:16D-PX which represents one bull. Samples
were collected at 30-min intervals for 26 h
through a jugular cannula. Standard error
difference for photoperiod treatment was
44.0 ng/ml of serum and for age was 27.8 ng/
ml of serum.

94

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95

Testosterone in plasma was unaffected (P > .25) by
photoperiod but at wk 0 (20 wk of age) tended to be greater
(P = .14) in SPX (1.57 ng/ml) than in PX (.67 nd/ml) calves
(figure 15). However, this divergence was not maintained
and testosterone concentrations were not different (P > .25)
between surgical treatments at wk 4 and 8.

At wk 0, LB in plasma was numerically greater in PX
(6.1 ng/ml) vs SPX (4.1 ng/ml) calves but the difference was
not significant (P > .25). By wk 4, LH converged (P < .l)
to 2.9 and 3.2 ng/ml of plasma in PX and SPX calves.
Photoperiod treatment did not affect plasma concentrations
of LH.

Discussion

 

In cattle, increasing day length from 8 to 16 h per day
is positively correlated with PRL concentrations in blood
(Stanisiewski et al., 1984). Furthermore, the eye is
essential for mediating the photoperiod-induced PRL response
in cattle (Petitclerc et al., 1983b). As previously
described, photic signals reaching the retina in most
mammals are transduced into neural signals which follow a
path through the suprachiasmatic nuclei (SCN) to the lateral
hypothalamus where sympathetic axons project to the upper
thoracic cord, through the superior cervical ganglia (SCG)
and finally terminate at the pineal gland (Reiter, 1983).
Following SCG removal (Lincoln et al., 1982) or pinealectomy
(Barrell and Lapwood, 1979) serum concentrations of PRL and

testosterone do not change in response to changes in

96

Figure 15.

Age-adjusted testosterone in plasma from PX
and SPX bull calves. Each observation re-
presents the mean of eight (SPX) or five
(PX) bulls where samples were collected at
30-min intervals for 26 h through a jugu1ar
cannula . Standard error difference for sur-
gical treatment was .27 ng/ml of serum and
for age was .13 ng/ml of serum.

97

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98

photoperiod in rams. In contrast, I demonstrated that
16L:8D increased PRL concentrations in PX as well as SPX
calves, although the response tended to be less in PX
calves. These results confirm those of Petitclerc et al.
(1983b) using more animals. Furthermore, both PX and SPX
calves receiving 16L:8D appeared to become refractory to
this photoperiod since PRL concentrations numerically
declined between 4 and 12 wk. This phenomenon has been '
shown previously in calves (Stanisiewski et al., 1987). In
cattle, except for the eyes, the essential components of the
pathway for photoperiod controlled PRL secretion are
unknown. However, based on current evidence, the pineal
gland in cattle may not be essential for transduction of
photic signals to the anterior pituitary gland.

Photoperiod can modulate puberty onset in a variety of
species including heifers (Petitclerc et al., 1983a), boars
(Mahone et a1., 1979), rams (Howles et al., 1980) and deer
(Budde, 1983). For example, 14 wk-old peripubertal-bulls
exposed to 16L:8D have higher testoSterone concentrations in
serum than controls receiving 8L:16D (Stanisiewski et al.,
1987b) suggesting a photoperiod effect on puberty. In the
present study, however, photoperiod did not affect
testosterone concentrations. The reason for this
discrepancy is unknown, although calves in the present study
were approximately 12 wk older at the start of photoperiod
treatment than in the previous study. This age difference
may be important if hull calves are only photoreceptive, in

terms of puberty onset, prior to 20 wk of age.

99

Testosterone concentrations normally increase in bulls
between 8 and 24 wk of age (Bedair and Thibier, 1979: Amann
and Walker, 1983). Relative to testosterone concentrations
in SPX calves in the present study. pinealectomy tended to
delay the increase. Based on concentrations of testosterone
in blood of l4—wk old calves (Stanisiewski et al., 1987b),
it appears that photoperiod affects puberty onset in bulls,
and at least during part of the peripubertal cascade of
hormonal events (McCarthy et al., 1979b) the pineal gland
may be involved.

In agreement with previous reports in cattle (Bourne
and Tucker, 1979; Rzepkowski et al., 1982), concentrations
of LH were not affected by photoperiod and therefore are not
a likely mediator of photoperiod or pineal-regulated
testosterone secretion. Species such as hamsters, sheep and
goats are seasonal breeders and rely heavily on an accurate
photoperiod evaluation system so that reproductive
capabilities are maximized during optimal times of the year.
In seasonal breeders, the photoperiod evaluation system
appears to consist of neural connections between the retina,
hypothalamus and pineal gland. Cattle, on the other hand,
are non-seasonal breeders and may have different
requirements for photoperiodic regulation of hormone
secretion, and the pineal gland is not involved in the
mechanism with photoperiod. Perhaps this explains why the
pineal gland of cattle is not essential for mediation of

photoperiod-induced changes in secretion of PRL and why LH

is unaffected by photoperiod.
100

In cattle, as is the case for all mammals examined so
far (Vollrath, 1981), melatonin concentrations in blood are
greatest in response to darkness as compared with exposure
to light. Pinealectomy abolishes nocturnal increases of
melatonin but does not completely eliminate the hormone from
blood (Cardinali, 1981: Bittman et al., 1983) and my study
confirms these results. Although the pineal gland is the
predominant source of melatonin, and the source of
essentially all of the nocturnally-secreted increment in
melatonin (Goldman and Darrow, 1983), other tissues can
synthesize melatonin (Rinkley et al.. 1979: Bubenik, 1980).
Extrapineal sources could account for the chronic presence
of melatonin in our PX calves. In addition, other evidence
suggests that baseline melatonin in blood of sheep increases
with time after pinealectomy (Kennaway et al., 1977) and
perhaps in the absence of the pineal gland, other tissues
increase production of melatonin. It is also interesting to
note that partial removal of pineal tissue in three of my
calves did not appear to diminish nocturnal melatonin
responses, which suggests pineal regeneration or
compensatory secretion by the remaining tissue.

In several species in which photoperiod regulates
anterior pituitary hormone secretion via melatonin output
from the pineal gland, duration of elevated melatonin is the
maior regulator (Goldman et al., 1984: Yellon et al., 1985)
rather than amplitude or total output of melatonin. In

fact, a 16 h elevation of melatonin in blood of sheep.

101

induced by feeding or iniecting melatonin, decreases PRL in
blood relative to 16L:80 photoperiod exposure (Kennaway et
a1., 1982, 1982/83). However, because 16L:8D stimulated PRL
concentrations in my PX calves without a concomitant
nocturnal melatonin rise, I conclude that diurnal melatonin
changes are not important in the regulation of photoperiod-
induced PRL release in cattle.

Bovine pineal tissue also contains specific PRL
releasing and PRL inhibiting factors (Blask et al., 1976;
Vriend et al., 1980). Whether these pineal factors play a
physiological role in photoperiod-induced PRL control is
unknown.

I conclude that the pineal gland is not an essential
mediator of changes in photoperiod-induced PRL
concentrations but may be involved with prepubertal

testosterone changes in bull calves.

Chapter 5

Melatonin and Prolactin Concentrations in Serum of Bull

Calves Exposed to 8, 16 or 24 Hours of Daily Light

103

Introduction

 

Melatonin is synthesized by the pineal gland of
vertebrate species in a circadian pattern with
concentrations being greatest during darkness and lowest
during periods of light (Wurtman et al., 1969: Cardinali ,
1981). Melatonin is also produced by extrapineal gland
sources, including gut (Bubenik, 1980), retina (Ralph, 1981)
and Harderian gland (Cardinali and Wurtman, 1972), and this
may account for the inability of pinealectomy to abolish
completely circulating melatonin concentrations (Bittman et
a1., 1983; Cardinali, 1981). Nevertheless, the pineal gland
remains the primary source of nocturnal increments in serum
melatonin (Arendt et al., 1980: Bittman et a1., 1983).

In seasonal breeding species such as sheep and
hamsters, the secretory pattern of melatonin acts as a
transducer between photoperiod and secretion of
gonadotropins and PRL (Bittman and Karsch, 1984; Goldman and
Darrow, 1983: Kennaway et al., 1982/83). In cattle, a non-
seasonal breeding species, long-day photoperiods (16L:8D)
consistently increase PRL concentrations in serum relative
to those in animals exposed to short days (8L:16D)
(Stanisiewski et a1., 1984; Tucker et al., 1984). However,
pinealectomy does not abolish the ability of photoperiod to
alter serum concentrations of PRL in cattle (Petitclerc et
al., 1983b: Stanisiewski et al., 1987a). My objective was
to characterize melatonin concentrations in blood collected

in calves exposed to 8, 16 or 24-h of daily light and to

104

determine whether daily administration of melatonin for 8 or
16 h affects PRL secretion in calves with or without pineal
glands.

Materials and Methods

 

Pretreatment. Holstein bull calves were used in three

 

experiments (eight per experiment) and were initially housed
in a light-controlled room from approximately 3 d of age and
received 8L:16D daily. For Exp. 1 and 2, calves were
subsequently moved into one of two light- and temperature-
controlled (16.6 i 1.3 C) chambers (Stanisiewski et al.,
1984) at 14 wk of age and maintained under 8L:16D for 3
additional wk. There were four calves per chamber.

In Exp. 3, two calves were pinealectomized (PX) and
four sham pinealectomized (SPX) between 6 and 7 wk of age.
The PX surgical procedure followed was that of Braun et al.
(1977). PX and SPX surgeries were performed identically
except in SPX calves the pineal gland was identified but
left intact. After surgery calves were moved into the
light- and temperature-controlled (23.4 i 1.5 C) chambers at
18 i 3 (X : S.E.) wk of age. All calves in Exp. 3 received
continuous light (24L:OD) from 13 wk of age.

Treatments and Blood Collection Exp. 1. On the last

 

day of pretreatment (d 1 of treatment). calves were fitted
with indwelling iugular catheters coupled to a 3 m cannula
which was passed into an adiourning room where blood was

collected remotely without disturbing the animals. Blood

was collected and discarded for l h to establish proper

105

cannula function. Beginning at 1200 h, blood was collected
(initial bleeding) for 24 h (d l) at 30-min intervals and at
2-h intervals for an additional 24 h (d 2). On the day
after initial blood collection, photoperiod was switched to
16L:8D in one chamber while the remaining four calves in the
other chamber were maintained on 8L:16D. After 4 wk of
photoperiod treatment calves were catheterized and bled
again (final bleeding) as described. One calf receiving
8L:16D died during the experiment, and all data from this
calf were discarded.

Exp. 2. Housing, duration of treatments and blood
sampling procedures were identical to those of Exp. 1.
However, photoperiod treatments were 24L:OD (four calves)
and 8L:160 (four calves).

Exp. 3. A11 calves were maintained under 24L:OD for
the duration of treatment. Approximately 3 wk after being
moved into the light- and temperature-controlled chambers,
each calf was fitted with a iugular cannula connected to an
infusion pump. Melatonin in saline was infused at a rate of

60.7 ug/h'50 kg'1

body weight for 16 h (pumps on at 1700 h).
The dose of melatonin was similar to that used in sheep to
mimic nocturnal concentrations in serum (Bittman et a1.,
1983). After 4 wk of 16 h daily melatonin infusion, the
contralateral iugular of each calf was catheterized and
blood was collected (initial bleeding) starting at 0800 h

for 25 h at 30-min intervals. On the following day, one PX

and two SPX calves were switched to an 8 h infusion regimen

106

(pumps on at 1700 h) while the remaining PX and SPX calves
were maintained on 16 h daily infusion for 5 additional wk.
On the final day of the experiment, blood was collected
again (final bleeding) for 25 h as described.

Blood Handling and Radioimmunoassays. Blood samples
were allowed to clot for l to 2 h at room temperature, then
stored for 24 h at 5 C. Samples were centrifuged for 20 min
at 1000 x g, decanted, and serum stored at -20 C until
assayed.

Prolactin was quantified in serum as reported
previously (Koprowski and Tucker, 1971). Direct assay of
serum concentrations of melatonin were performed using the
procedures and antibody of Fraser et al. (1983). Validation
of the melatonin assay has been reported (Stanisiewski et
al., 1987a).

Statistical Analysis. Split-plot analysis of variance
(Gill, 1978b) was used to test for hormone concentration
differences within and among treatments in Exp. 1 and 2. A
2 X 2 factorial design with repeat measure was used in Exp.
3. See appendix for a listing of sources of variation and
specific contrasts.

Results

Exp. 1, Prolactin. After 17 wk exposure to 8L:16D, PRL
averaged 6.3 and 3.8 ng/ml of serum (P > .25) on d l of .
treatment for the two groups of calves. In calves
subsequently switched to 16L:8D for 4 wk, PRL increased (P <

.05) to 30.9 ng/ml of serum compared with 7.0 nq/ml in

107

;( ‘l'l

 

 

calves maintained on 8L:16D. Concentrations of PRL did not
vary diurnally.

Melatonin. Serum concentrations of melatonin on d 2

 

were similar to those on d l, and since blood was collected
more frequently on d l, statistical analysis of d 1 values
will be described. Melatonin during darkness averaged 41.8
and 32.4 pq/ml of serum (P < .25) in the two groups of
calves at initial bleeding (8L:16D). With lights on,
melatonin decreased (P < .01) to an average of 13.9 and 20.2
pg/ml of serum (P > .25) in each group. One calf had
persistently high melatonin in blood throughout d 1 and 2
and was eliminated from initial bleed calculations after
determination (procedure of Grubbs and Beck) that he was an
outlier (P < .05: Gill, 1978a). At final bleeding,
nocturnal melatonin averaged 60.1 and 35.0 ng/ml of serum (P
< .05) in 16L:8D and 8L:16D treatments, respectively (figure
l6, 17). With lights on, melatonin decreased (P < .05) to
an average of 11.9 (16L:8D) and 9.6 (8L:16D) Dq/ml of serum
(P > .25: figure 16, 17).

During initial bleeding, melatonin expressed as total
area under the curve was greater (P < .01) during darkness
(40.9 and 31.3 nq'min'ml—l: P > .25) in each group of calves
compared with values during the light period (5.8 and 9.2

ng'min'ml-I: P > .25) At final bleeding, nocturnal

melatonin areas (27.0 and 34.0 ng'min'ml—l: P < .1) remained

greater (P < .01) than areas during light exposure (10.3 and

4.2 ng'min'ml_1: P < .05) in 16L:8D and 8L:16D treatments,

108

;: till

Figure 16.

Forty-eight h profiles of melatonin in serum
of four bull calves receiving 16L:8D after 4
wk on experiment. Samples were collected
through a jugular cannula at 30—min intervals
for the first 24 h and at 2 h intervals‘
thereafter. The hatched bar along the x-
axis represents darkness. Standard errors
of means during light and darkness: respect-
ively, were 8.2 and 27.3 pg/ml of serum.

109

 

 

 

 

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Figure 17.

Forty-eight h profiles of melatonin in serum
of three bull calves receiving 8L:16D after
4 wk on experiment. Samples were collected
through a jugular cannula at 30-min intervals
for the first 24 h and at 2 h intervals
thereafter. The hatched bar along the x-
axis represents darkness. Standard errors
of means during light and darkness; respect-
ively, were 6.6 and 18.0 pQ/ml of serum.

111

 

 

 

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112

respectively. Total melatonin areas per 24 h were not
different between 8L:16D vs 16L:8D (P > .25)

When darkness was administered for 16 h at initial and
final (figure 17) bleedings, melatonin declined to baseline
concentrations 2 to 4 h prior to lights on: in other words,
the nocturnal melatonin increment lasted 12 to 14 h. In
calves receiving 8 h of darkness at final bleeding,
melatonin concentrations were elevated for the entire
duration of dark exposure: i.e. 8 h (figure 16), but was
suppressed with onset of lighting.

Great variability existed among individual calves both
in terms of pulsatile activity and magnitude of the
nocturnally-induced melatonin increment (figure 18).
Highest daily concentrations of melatonin for individual
calves always occurred in response to darkness and ranged
from 34.0 to 291.0 pg/ml of serum. In contrast, highest
melatonin values with lights on were 9.6 to 57.2 pg/ml of
serum.

Exp. 2, Prolactin. After 17 wk exposure to 8L:16D, PRL

 

averaged 11.0 and 11.3 ng/ml of serum (P > .25) on d l for
each group of calves. Prolactin in serum was unchanged (P >
.25) 4 wk later in response to either 24L:OD or to
continuation of 8L:160 (12.9 vs 15.6 ng/ml: P > .25).

Melatonin. At initial bleeding, mean melatonin was

 

265% greater (P < .01) during the dark (30.7 pg/ml of serum)
period as compared with the light (8.4 pg/ml) period in each

group of calves exposed to 8L:16D. In three calves

113

'1 [‘1‘

 

 

 

". {:11

Figure 18.

Twentyufour h profiles of melatonin in serum
of two bull calves receiving 8L:16D. Samples
were collected at 30—min intervals through
a jugular cannula. The hatched bar along
the x-axis represents darkness. Standard
errors of means during light and darkness:
respectively, were 2.5 and 42.0 pg/ml of
serum for calf 8163 and 7.4 and 19.6 pg/ml
for calf 8133.

 

 

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115

'1 till

 

receiving 24L:OD, melatonin averaged 9.3 pq/ml of serum over
24 h and there was no nocturnal increase in melatonin.
However, in one calf subsequently switched to 24L:OD,
melatonin increased during a period corresponding to light
exposure 4 wk earlier (figure 19). In calves maintained on
8L:16D melatonin averaged 8.0 pg/ml during light and 39.7
pg/ml of serum during darkness and the pattern was similar
to that in Exp. 1 (figure 17). Concentrations of melatonin
in all treatments and in all calves were similar (P >.25)
between bleedings on d l and d 2.

Exp. 3, Prolactin. Surgical treatment did not affect
(P > .25) concentrations of PRL in serum, therefore PX and
SPX data were pooled within each infusion regimen for
further analysis. After 4 wk of 16 h daily melatonin
infusion, PRL averaged 22.7 and 20.0 ng/ml of serum in each
group of calves (P > .25). Five wk later, PRL was similar
(P > .25) in calves switched to an 8 h infusion of melatonin
(21.0 ng/ml of serum) as compared with calves infused with
melatonin for 16 h per day (20.8 ng/ml).

Melatonin. Concentrations of melatonin in serum were
quantified only during final bleeding to ensure that
indwelling infusion cannulas continued to function pronerly.
Within 1 to 3 h of infusion onset, melatonin increased in
serum (figure 20, 21). In serum melatonin averaged 637.8
pg/ml during the 8 h infusion and decreased (P < .01) to an
average of 88.9 pg/ml during the 16-h interval when bulls

were not infused. In calves infused for 16 h, melatonin

116

'11:”

Figure 19.

Twenty-four h profiles of melatonin in serum
of three (8164, 8170, 8171) or one (8166)
bull calves receiving 24L:0D. Samples were
collected at 30-min intervals through a
jugular cannula. Standard errors of means
were 1.6 pg/ml of serum for calves 8164,

8170 and 8171 and 12.7 pg/ml for calf 8166.

117

 

 

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118

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Figure 20.

Twenty-four h profiles of melatonin in serum
of three bull calves receiving 24L:OD and
infused intravenously with melatonin for 8 h
(hatched bar). Standard errors of means
during non-infusion were 26.6 pg/ml of
serum and 318.9 pg/ml during infusion.

119

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120

‘x‘hl;

Figure 21.

Twenty-four h profiles of melatonin in serum
of three bull calves receiving 24L:OD and
infused intravenously with melatonin for 16 h
(hatched bar). Standard errors of means
during non-infusion were 6.9 pg/ml of
serum and 141.8 pg/ml during infusion.

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122

averaged 565.0 pg/ml of serum and decreased (P < .01) to an
average of 31.4 pg/ml during the 8-h interval when not
infused.

Discussion

 

In agreement with results in other vertebrates
(Cardinali, 1981), I found that circulating melatonin
increases in response to darkness in calves. I also
expanded on the results of Hedlund et al. (1977) and Martin
et al. (1983), who demonstrated a 6- to 7-fold nocturnal
rise of serum melatonin in cattle, to show that duration of
the nocturnal increment is altered with duration of
darkness. Through frequent (30-min interval) sampling,
marked pulsatile changes were.observed in concentrations of
melatonin in calf serum. Bittman et a1. (1983) also
suggested that melatonin is released episodically in sheep.

Stanisiewski et al. (1984) previously demonstrated that
relative to 8L:16D, PRL concentrations in serum of bull
calves increase in response to 16L:8D exposure. In
addition, previous results were confirmed (Stanisiewski et
al., 1987c) that calves receiving continuous light for less
than 6 wk have serum PRL concentrations that do not differ
from those in calves exposed to 8L:16D. Similar to results
in cattle, PRL concentrations in sheep are greater in
response to 16L:8D exposure than to 8L:16D (Pelletier,
1973). Pinealectomy of rams (Barrell and Lapwood, 1979),
ewes (Munro et a1., 1980) or white-tailed deer bucks

(Schulte et al., 1981), abolishes or greatly reduces

123

photoperiod-induced PRL rhythms, thus implicating melatonin
or some other pineal gland product as a regulator of
photoperiod-induced PRL secretion. In fact, afternoon
feeding of melatonin to sheep or deer (Kennaway et al.,
1982; Webster and Barrell, 1985) or injecting ewes (Kennaway
et a1., 1982/83) with melatonin reduces concentrations of
PRL in blood similar to that in animals receiving short-day
photoperiods. In contrast, pinealectomy of cattle has
little if any effect on photoperiod-induced PRL
concentrations (Petitclerc et al., 1983b; Stanisiewski et
al., 1987a). When comparing 16L:8D vs 8L:16D treatments in
my present study, daily mean serum concentrations of
melatonin as well as area under the curve per 24 h were
equivalent yet mean PRL concentrations were different. On
the other hand, daily mean concentrations of melatonin were
greater in calves exposed to 8L:16D than in calves receiving
24L:OD, yet, PRL concentrations were the same. It appears
from my results that mean daily concentrations of melatonin
have no correlation with mean daily concentrations of PRL in
prepubertal bull calves.

As stated, duration of increased nocturnal melatonin
concentrations in serum coincided with the duration of
darkness that calves received, and mean nocturnal melatonin
concentrations tended to be greater with shorter durations
of dark exposure. This was similar to data in rams where
mean nocturnal melatonin concentrations are greater in

animals exposed to long days compared with short days

124

(Lincoln et al., 1982). I successfully mimicked this
pattern in our melatonin-infused calves (Exp. 3). Although
melatonin concentrations in serum of our infused animals
were approximately 15-fold greater than average nocturnal
concentrations in non-infused calves in Exp. 1 and 2, these
infusion-induced concentrations achieved our goal of
matching reported values in melatonin-infused sheep (Bittman
et al., 1983). In 2-yr old steers, we observed nocturnal
melatonin above 400 pg/ml of serum (Stanisiewski and Tucker,
unpublished data). Therefore, I believe that infused
concentrations in the present study approximate those which
cattle may normally achieve. Since altering duration of
melatonin exposure by infusion (Exp. 3) did not affect
circulating PRL concentrations, it appears that duration of
elevated melatonin in cattle, unlike sheep (Bittman and
Karsch, 1984), is not a major controlling factor of
photoperiod-induced PRL release. In sheep (Bittman and
Karsch, 1984) and hamsters (Goldman et al., 1984), duration
of elevated melatonin concentrations control reproductive
and hormonal responses to melatonin. However, melatonin
secretion in cattle has not been definitively linked with
reproductive function or secretion of other hormones such as
PRL. Therefore, I cannot yet determine the functional
importance of factors such as duration, episodic release.
amplitude, total amount secreted or temporal phase (relative
to ambient light-dark exposure) of melatonin secretion in

cattle. I speculate that lack of control of the pineal

125

gland over PRL release in cattle may represent a divergence
of non-photoperiodic from photoperiod-regulated breeders.
This is also the first report in cattle demonstrating
that continuous light suppresses concentrations of melatonin
throughout a day. Similar results were reported in sheep
(Kennaway et al., 1983: Rollaq and Niswender, 1976), rhesus
monkeys (Perlow et al., 1980) and rats (Ralph et al., 1971).
However, I cannot explain the diurnal melatonin pattern in
one calf receiving 24L:OD. This pattern (detected on
consecutive days) was 180 degrees out of phase with the
8L:16D-induced pattern of 4 wk earlier. Humans are the only
species known to have melatonin rhythms which persist in
constant illumination (Vaughan et al., 1976: Weitzman et
al., 1978). However, this phenomenon in humans may be a
result of exposure to insufficient light intensity to
suppress endogenous melatonin rhythms. For example, in
Syrian hamsters, 1 1x is sufficient to suppress melatonin
(Reiter, 1983) while in ewes (Kennaway et a1., 1983), 50 1x
is sufficient. Humans, on the other hand, require 1500 to
2500 lx (Lewy et al., 1980). Whether the light intensity
used in the present study was only marginally adequate to
suppress melatonin in calves is unknown. Another
possibility is that calves may go through a natural
progression in which diurnal melatonin release continues
upon removal of darkness, and this diurnal pattern
eventually decreases and (or) phase shifts. Perhaps this

had not yet happened in the one calf.

126

In summary, blood collection intervals of 2 h are
adequate for characterization of nocturnal melatonin
duration. Elevated melatonin concentrations follow duration
of darkness and constant illumination eliminates the diurnal
pattern. Prolactin in serum is greater in calves receiving
16L:8D than in 8L:160 exposed animals, however, altering
increments of melatonin by infusion of melatonin does not
affect PRL. I conclude that diurnal melatonin secretion
probably does not mediate photoperiod-induced changes in

serum PRL concentrations in prepubertal bulls.

127

SUMMARY AND CONCLUS IONS

Photoperiod has marked effects on PRL concentrations in
serum of cattle. Namely, relative to 8L:16D, 16L:8D
increases PRL concentrations in blood but the increased
level is not maintained indefinitely. .On the other hand,
8L:16D maintains relatively constant PRL concentrations in
calves for at least 12 wk, provided ambient temperature is
constant. Abruptly switching from 8L:16D to continuous
lighting will also increase PRL in blood, but the time
course of the response is delayed with respect to 16L:80.

In addition, serum testosterone concentrations increase
at an earlier age in young bulls receiving 16L:8D compared
with bulls receiving 8L:16D. Furthermore, this response is
not synchronous with changes in mean concentrations or
pulses of LH. Also, concentrations of PRL are increased
equally in castrated and intact male calves switched from 8
to 16 h of light per day. Therefore, the gonads are not
required for regulation of photoperiod-induced PRL
concentrations. However, since LH concentrations are
unaffected by photoperiod, the photic response on the gonad
may be a result of PRL action alone or in synergy with basal
LH concentrations.

The neuroendocrine mechanism whereby light affects PRL
release in cattle remains a mystery, although the PRL

inhibitor, DA, may be involved. For example, concentrations

128

of DA were higher in pituitary glands from calves receiving
8L:16D as compared with concentrations in glands from
16L:8D-exposed calves. In addition, since PX does not
abolish 16L:8D-induced PRL concentrations, the pineal gland
is not necessary for photic signal transduction in cattle.
The lack of requirement for the pineal gland to relay
photoperiod-controlled PRL secretion in cattle is in
contrast with other species such as hamsters and sheep, and
may represent an evolutionary divergence of non-
photoperiodic and photoperiod-regulated breeders. One
function the pineal gland may serve in cattle is on
regulation of puberty onset, since concentrations of
testosterone were lower at 20 wk of age in PX bulls compared
with SPX bulls, but again, LH concentrations were not
different between the two groups.

Mean concentrations and diurnal rhythmicity of
melatonin are altered by photoperiod in calves. Melatonin
is secreted in pulses, and circulating concentrations are
greatest during darkness. Furthermore, providing 8 h of
darkness results in melatonin concentrations being elevated
for 8 h whereas melatonin is high for only the first 12 to
14 h of a 16 h dark period. In addition, melatonin is
suppressed to basal (daylight) concentrations when
continuous daily light is provided. I showed that
concentrations of PRL in serum of calves can be the same in
spite of markedly different patterns and concentrations of

melatonin. Infusion of melatonin for 8 or 16 h does not

129

affect PRL concentrations in calves. Since melatonin is a
primary hormone secreted from the pineal gland, the lack of
melatonin effect on PRL is further evidence of the
dissociation of the pineal gland from photoperiod-induced
PRL regulation in cattle.

It is concluded that although long-day photoperiod
exposure increases serum concentrations of PRL in bull
calves, the mechanism does not appear to involve the pineal
gland or melatonin secretion. However, DA appears to be a
photoperiod-regulated neurohormone which may at least
partially be responsible for photoperiod-induced PRL
release. Furthermore, PRL concentrations in bull calves
become refractory to prolonged long-day exposures.

The mechanism through which long-days increase
testosterone concentrations at an earlier age in bulls
appears to be dependent upon the pineal gland, and may be a
result of synergism of PRL and (or) pineal secretions on

basal concentrations of LH.

130

.=JJI:"

 

 

APPENDIX

APPENDIX

Experimental models depicting sources of variation and
degrees of freedom for experiments contained within each
chapter. Animals excluded from analysis were treated as

missing data.

 

 

 

Chapter 1. Exp. 1: Source of Variation g:
Photoperiod Treatment(P) l

Calves/P 6

Day of Bleeding (B) 3

P x B 3

Calves/P x B 1 18

Time of Blood Collection(T) 52

P x T 52

B x T 156

P x B x T 156

Calves/P x B x T 1009

Total = 1456

Exp. 2: Source of Variation g:
P 1

Calves/P 6

B 2

P x B 2

Calves/P x B 12

T 12

P x T 12

B x T 24

P x B x T 24

Calves/P x B x T 143

Total = 238

Chapter 2. Exp. 1: Source of Variation g:
P 1

Calves/P 6

B 2

P x B 2

1When not significant (see text). T was deleted from models.

131

Exp. 2

Chapter 3.

Chapter 4.

2

Calves/P x B 12

 

 

 

T 12
P x T 12
B x T 24
P x B x T 24
Calves/P x B x T 304
Total = 29
Source of Variation g:
Effect of Castration(C) l
Calves/C 6
B l
C x B l
Calves/C x B 6
T 12
C x T 12
B x T 12
C x B x T 12
Calves/C x B x T 144
Total = 2 7
Sources of Variation g:
P 1
Calves/P 6
B l
P x B l
Calves/P x B 6
T 12
P x T 12
B x T 12
P x B x T 12
Calves/P x B x T 193
Total = 16
Source of Variation ‘g:
Replicate (9)2 l
Effect of Pinealectomy(S) 1
P l
S x P . 1
R x S 1
R x P 1
R x S x P 1
Covariate 1
Calves/R x S x P 7
B 3

Not significant for testosterone, LH or PRL (when adjusted

or ambient temperatures).
Covariate = Age(testosterone, LB) or Ambient temperature

132

 

 

S x B 3
P x B 3
S x P x B 3
R x B 3
R x S x B 3
R x P x B 3
R x S x P x B 3
Covariate 1
Calves/R x S x P x B 23
Total = 63

Chapter 5. Exp. 1, 2: Source of Variation g:
P l
Calves/P 6
B l
P x B l
Calves/P x B 6
Presence/Absence of Light(L) 1
P x L l
Calves/P x L 6
B x L l
P x B x L l
Calves/P x B x L 21
Total = 52

Exp. 3 Source of Variation g:
S 1
Melatonin Infusion (I) 1
S x I l
Calves/S x I 2
B 1
S x B l
I x B 1
S x I x B l
Calves/S x I x B 357

*3
O
('1’
0)
p...
ll
0
0‘
Ch

133

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