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THE INABILITY TO PERCEIVE PHOTOPERIOD AFFECTS

REPRODUCTION IN HINK

BY

Katherine A. Koudele

‘A DISSERIATION
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

1988

ABSTRACT
THE INABILITY TO PERQEIVE PHOTOPERIOD AFFECTS
REPRODUCTION IN HINK
BY

Katherine A. Koudele

The predictable changes in photoperiod throughout the
year stimulate changes of reproductive function in most
non-primate animals. In mammals, the physiological pathway
for photoperiod detection in mammals begins with the eyes,
then to the superior cervical ganglia (SCG) which relays
the information concerning daylength on to the pineal gland.
If the eye-SCG-pineal axis is interrupted, disturbances in
reproductive function result.

To separate the roles of the eyes and the SCG in
photoperiod- controlled reproduction, 72 prepubertal (6
months old) mink (36 male and 36 female) received one of
the following surgical treatments in November or December
of their first year: 1) bilateral blinding, 2) bilateral
superior cervical ganglionectomy (SCGx), 3) blinding +
SCGx, 4) blinding + sham SCGx, 5) sham SCGx, or 6) remained
intact. The animals remained under the naturally-occurring
photoperiod in East Lansing, Michigan, although one-half of
each surgical treatment group was housed indoors or
outdoors and both under standard mink ranch conditions.
The animals were examined every two weeks from one month

post-surgery (January) through two' breeding seasons

(March). Body weight was recorded and reproductive
development assessed by testicular dimensions or degree of
vulvular swelling. Blood samples were collected by jugular
venipuncture and the serum from males was analyzed for
testosterone (T) and serum from females for estradiol (E2).

The pubertal increase in testes size, vulva edema, T
.or E2 concentrations did not differ among treatment groups.
However, all three blinded mink groups maintained enlarged
testes or vulvae significantly longer after the breeding
season than sighted mink. Testes involution in blinded
males was slower than sighted males with the testes not
ascending into the abdomen until July while the sighted
males testes ascended in May. The T profile paralleled and
slightly preceded that of the testes size in all groups.
Testes recrudesence of SCGx males began in November, one
month before sham SCGx or intact males. Blinded males
showed no recrudesence by the second breeding season. The
E2 profiles were too varied within the treatment groups to
yield any conclusive results about the effect of the
surgeries on E2 levels. The blinded animals of both sexes
exhibited no circannual changes in body weight as did the
sighted animals. There was no effect of housing on any
reproductive parameters measured.

It appears that the eyes are the most crucial component
in the eye-SCG-pineal axis in mink since without them the

animals become asynchronous with the photoperiod despite

other treatments. There was no spontaneous regeneration of
the gonads and the post-breeding season atrophy of the
testes was slower in blinded animals indicating that mink
require the eyes to detect the photoperiod not only to

stimulate reproductive function but to terminate it as well.

ACKNOWLEDGEMENTS

I extend my thanks first to my major professor, Dr.
Richard Aulerich, for accepting this virtual orphan into
his research program. He guided and supported me as I
began an entirely new research project and have now
completed it. His confidence in me, enthusiasm, and common
3 ense helped keep me going when I became discouraged. His
willing and competent assistance in the "hands-on" portion
of my research is greatly appreciated. I feel very
fortunate to have had the priviledge of having him as my
advisor. .

I would also like to thank my other committee members,
Drs. Roy Fogwell, James Asher, and Lynwood Clemens, for
their enthusiasm and support of my research. They provided
me with a number of creative ideas to my research problems.

And lastly, I want to thank past and present members of
the Animal Reproduction Lab for permitting me to continue
to work in their lab and for their support of my research.
My sojourn here has been a very good five years of my life,
and most of that is due to the personal friendships I have

made with them.

TABLE OF CONTENTS

’ Page
List or Tables 0.00.00.00.0000000000000000000000000....O. Viii
List Of Figures OOOOOOOOOOOOOOOOOOOOOO .......... O ...... .0 ix
Review of Literature

100 IntrOductionOOOOOOOO0.0.0.0....0...I. ........... 1

2.0 Photoperiodicity and Reproduction
inuammals OOOIOOOOOOOOOOO0.0.0.0000... ......... 2
2.1 Investigations of Wildlife Species.......... 2
2.2 Investigations of Laboratory Species........ 4
2.3 Investigations of Large Domestic Species.... 6
2.4 Investigations of Mink and Ferrets.......... 7

3.0 Physiology of Photoperiodicity.................. 12
3.1 Introduction................................ 12
3.2 The Pineal Gland............................ 13
3.2.1 Brief Description of Morphology....... 13
3.2.2 Control of Melatonin Synthesis........ 16
3.2.3 Effects of Melatonin and Pinealectomy
on Reproduction....................... 19
.3 Interaction of Eyes with Pineal Gland ...... 24
.4 Interaction of Hypothalamus with Pineal
Gland........................................ 27
4.0 Photoperiodic Control of Puberty and Recurring
Cycles.......................................... 29
4.1 Neural Input and Pubertal Processes......... 29
4.2 Involvement of the Pineal in the Photoperiodic
Initiation of Puberty....................... 31
5.0 Concluding Remarks.............................. 35

UN

IntrOduction COOOOOOOOOCOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00 36

Materials and Methods
0bjective........................................... 38
Specific Hypothesis................................. 38
Statistical Design.................................. 38
Animal Care......................................... 42
General Surgical Procedures......................... 42
Specific Surgical Procedures........................ 43
Blood Collection Procedure.......................... 46
Physical Measurements .............................. 47
Reproductive Behavior Evaluations................... 48
Radioimmunoassay Procedures................... ...... 49

vi

Results
Body Weight........................... ....... .......
External Genitalia .................................
Steriod Hormones....................................
Hair Coat...........................................
Reproductive Behavior.................. ....... . .....

DiscuSSionOOOOOO0.0.0.0....OOOOOOOOOOOOOOOOOOOOOOOOOOO...
conCIuSionSOOOIOOOOO0.00...OOOOOOOOOOOOOOOOOOOOOO ........

List Of ReferenceSOOO'OOOOOOOOOOOOOOOOOOOOOOOOOO0.... .....

vii

51
60
60
69
50

79

86

87

LIST OF TABLES

Main statistical design of a double split
plot with sex effect analyzed separately.

Design for analyzing unbalanced data for
females by surgical treatment and housing.

viii

39

40

10

11

12

13

14

LIST OF FIGURES

Weekly changes in photoperiod from

January

1 through June 24 at the 45th N

parallel (calcuated from nautical almanac)

and the

reproductive cycle of mink.

Presumed components of the photoperiod

sensing

and processing system in the mink

brain (based on hamster data).

Changes

in body weight over time in sighted

male mink across housing type.

Changes

in body weight over time in blinded

male mink across housing type.

Changes

in body weight over time in sighted

female mink across housing type.

Changes

in body weight over time in blinded

female mink across housing type.

Changes

in testicular volume over time in

sighted male mink across housing.

Changes

in testicular volume over time in

blinded male mink across housing.

Changes

in vulvar edema over time in sighted

female mink across housing.

Changes

in vulvar edema over time in blinded

female mink across housing.

Changes
time in

Changes
time in

Changes
time in

Changes
time in

in testosterone concentrations over
sighted male mink across housing.

in testosterone concentrations over
blinded male mink across housing.

in estradiol concentrations over
sighted female mink across housing.

in estradiol concentrations over
blinded female mink across housing.

ix

14

52

54

56

58

61

63

65

67

70

72

7'4

76

1-0) Introduction

This discussion will focus on photoperiodicity and its
role in reproduction of mammals, mainly laboratory species
and some carnivores. This phenomenon is poorly understood
in carnivores although many are economically valuable
especially the family, Mustelidae.

A member of Mustelidae, the mink was the model species
for this dissertation, so more time than usual will be
spent discussing their reproductive response to
photoperiod, and that of a fellow mustelid, the ferret.

The physiological aspects of photoperiodicity will be
approached using the pineal gland as the primary
neurohormonal structure providing the biological signal
concerning length of night and day. The neural circuitry
to and from the pineal gland, as well as the hormonal
actions and reactions, will be discussed.

This literature review will also concentrate on an
aspect of reproduction that this research investigated, the
initiation of puberty and the annual onset of the breeding

season, and the influence of photoperiod upon it.

Most mammalian species reproduce during the time of
year when the probability for survival of both parents and
young is maximal (Turek and Campbell, 1979). In temperate
regions, the principle cue in triggering the cascade of
events which leads to successful parturition and rearing of
the young is the annual changes in photoperiod (Menaker,
1971). i

The grasshopper mouse inhabits the western United States
and breeds during the spring and summer months of long days
(March through August). Although the photoperiod is
increasing from March until the end of June and then
decreasing in July and August, it appears that it is the
total amount of light received in a day that is the
important cue. Ten hours of light, short Idays (SD), lead
to gonadal regression while 12 to 16 hours, long days (LD),
are sufficient to stimulate and maintain enlargement of
gonads. However, if the mice are housed under SD for 30
weeks for males or 16 weeks for females, they will undergo
spontaneous gonadal regrowth (Frost and Zucker, 1983).
Disparity in response between the sexes is not understood
although it has been noted in other rodent species as well.
This waning of the inhibitory effects of SD permits

late-born animals to be reproductively ready for the next

3
spring breeding season as soon as it arrives although the
daylight is not yet 14 hours long.

The white-footed mouse is also a long-day breeder and
when adult females are maintained on a schedule of 8 hours
of light and 16 hours of darkness (8L:16D) for 6 weeks,
their uterine and ovarian weights decrease markedly in
weight (Petterborg, 1983). However, a closely related
species, the prairie deer mouse, responds differently to
short days. Adult females on SD exhibit little disruption
of reproductive condition (Whitsett and Miller, 1982).

A relationship between prenatal exposure to
photoperiod and subsequent reproductive development has
been reported in voles (Horton, 1984, 1985: Nelson, 1985b).
Young voles respond to the length of daylight to which
their mothers were exposed during gestation, rather than to
the photoperiod that the young voles experienced from birth
until weaning. A similar response was discovered in the
Djungarian hamster by Stetson et al.(1986). This is
significant biologically since the young animals can begin
to prepare reproductively for the environment they are born
into so they will be ‘ready to breed when they are weaned.
However, Nelson (1985a) discovered that after a couple of
generations of laboratory breeding from wild-trapped
ancestors, the descendants have markedly reduced
' photoperiod sensitivity. Lab raised ground squirrels were
also less sensitive to light than wild squirrels (Reiter et

4
al., 1983). This phenomenon of reduced photoperiod
sensitivity after generations of laboratory breeding could
be an explanation for apparently conflicting results in
photoperiod responses of wild species.

Photoperiodicity has been investigated in species as
unusual as pallid bats found in Napa Valley, California.
The transition of long days to short days stimulates the
change in reproductive function from spermatogenesis and an
inability to copulate to sperm storage and the ability to
mate but with no sperm production (Beasley and Zucker,
1984). Large wild ungulates such as red deer (Webster and
Barrell, 1985) and white- tailed deer (Bubenik et al.,
1982), have been shown to respond to photoperiod,
specifically artificially produced short days, with early
antler growth, early mating and winter coat growth.

Most of the photoperiod work involving wild mammalian
species is descriptive with few of the controlled
experiments such as those reported using laboratory

species.

2-2) W

The hamster is to laboratory photoperiod studies what
the fruit fly has been to genetic research. The Syrian, or
golden, hamster has been the most commonly used species
although the Turkish and Djungarian hamsters have also been

studied.

5

The golden hamster is a seasonal breeder in which long
days are stimulatory to reproduction and short days
inhibitory. However, like the grasshopper mouse,
maintaining hamsters in short photoperiods (less than 12.5
hours light/24 hours) will not suppress gonadal growth
indefinately. After 20 to 25 weeks the animals will become
photorefractory and spontaneously regain reproductive
competence (Reiter, 1972; Stetson and Tate-Ostroff, 1981;
Steger et al., 1982). However, if they are kept in
greater than 12.5 hours light/24 hours, they undergo
—gonadal regression. The fact that the critical photoperiod
is 12.5 hours shows that the hamster is capable of fine
discrimination in daylength. When an animal has become
photorefractory on long days it requires an intervening
period of 10 to 11 weeks of short days to make it
"photosensitive" to long days again (Reiter, 1973a).

In a biological perspective the refractory phenomenon
can be understood. It occurs in nature from December
through February when reproductive success would be poor.
Spontaneous gonadal recrudescense begins in March and the
animals are reproductively competent and photosensitive to
the long days in May. This is also the best time to raise
offspring and have them survive.

The laboratory rat, not as photosensitive as the
hamster, has been selected for maximal reproduction

efficiency. It will continue to mate and raise litters

6
throughout the year if exposed to even a few hours of
light. The hamster may be headed toward the same fate since
photoperiod induced reproductive changes that occurred in 4
weeks in 1965 now take 6 to 8 weeks (Reiter, 1980a). If a
female rat is exposed to constant light, however, she will
go into a state of anovulation but with constant vaginal
and behavioral estrus (Lawton and Schwartz, 1967). Due to
their inability to respond to relatively small changes in
daylength, the lab rat is a poor model for photoperiod
studies. Their wild cousins, though, are probably as
photosensitive as any other wild animals. But in the wild,
other factors such as food availibility may override

photoperiodic effects on reproduction.

2-3) W

Much of the photoperiod manipulation in the environments
of large domestic (farm) animals has been done to increase
the food or fiber production by those animals (Tucker and
Ringer, 1982). This is accomplished either directly by
immediate increases in the amount of milk or other product,
or indirectly by controlling when reproduction will occur.
Although dairy cattle are not a highly photoperiodic
species in terms of reproduction, fertility is greater
during the long-day months of summer and fall than in the
winter (Tucker, 1982). Heifers reared on long days

(16L:8D) eat more and gain weight more rapidly than when

7
exposed to short days (8L:16D), consequently they reached a
larger body size and puberty sooner (Petitclerc et al.,
1983a).

The most commonly studied large animal species is the
sheep. Most breeds of sheep are photosensitive and the
decreasing daylength in the fall stimulates the onset of the
breeding season. Rams maintained under short days (8L:16D)
showed increases in testes size, and presumably fertility,
sooner than rams on long days (Almeida and Lincoln, 1982).
Ewes treated similarily followed the same pattern with an
earlier onset of estrus (Legan and Winans, 1981). However,
if the sheep are kept on short days for more than 16 weeks
the sexual competence wanes. and their gonads regress.
Conversely, if they are maintained under usually inhibitory
long days, the gonads spontaneously recrudesce. Sheep
appear to have a refractory mechanism as does the hamster

(Robinson and Karsch, 1984).

2-4) WWW
As stated in the introduction, the model species for

this research is the mink. Both mink and ferrets
mustelids breed during lengthening days although the testes
of the male mink begin to enlarge in decreasing daylength
(Bostrum et al., 1968; Nieschlag and Bieniek, 1975)
prompting some authors to call them ”short-day breeders"

(Boissin-Agasse et al., 1982) (Figure 1). The mink

Figure 1. Rate of increase of light per week from January 1
through June 24 at the 45th N parallel (calculated
from the nautical almanac) and the breeding cycle
of mink.

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10
breeding season begins about the first of March and lasts 3
to 4 weeks while that of the ferret starts in May and
continues for several months throughout the summer (Pilbeam
et al., 1979). Both species are induced-ovulators and the
mink displays the embryo diapause/delayed implantation
phenomenon (Hansson, 1947). Mink annual cycles of furring
and reproduction are closely tied to the photoperiod (Duby
and Travis, 1972).

Bissonnett (1932) discovered that ferrets can be
induced into early sexual competency by artificially
increased photoperiod in the late fall. Hansson published
the first detailed report in 1947 on reproductive
physiology in the mink. He tried unsuccessfully to hasten
the onset of the breeding season by exposing the animals
to an earlier dawn and a later dusk in the fall. He caused
only uterine and ovarian weights to increase. In 1951,
Hammond reported that exposing mink to artificially short
days (the actual photoperiod was not reported) in the summer
caused the females to exhibit estrus vaginal smears and the
males, enlarged testes. Ferrets exposed to 14 hours of
light in January experienced estrus earlier than those on
natural photoperiod or on constant light. Mink on the same
lighting regime did not exhibit estrus (Donovan et al.,
1983). Immature female ferrets begin to have ovarian
maturation within 22 days after being exposed to long days

at 16 weeks to age (Ryan, 1985). In general, ferrets

11
appear to respond reproductively to light manipulation more
quickly than do mink.

Female mink given extra lighting (8.2 minutes light
added/day) from February 1 showed estrus and willingness to
be mated earlier than females on normal photoperiod
(February 15 vs. March 5) (Holcomb et al., 1962).

Increased daylength before and after mating also reduced
gestation length and increased the number of kits whelped
per litter. Both these effects were due to decreased
embryonic diapause and earlier implantation. A later
attempt was make to produce two litters in the same year.
Daylength to the mothers was decreased after weaning (July
1) to that of the winter solstice by mid-July and then
gradually increased (Aulerich et al., 1963). The females'
vaginal smears were estrual in the fall but there were no
matings since the males showed little interest in mating.

Travis and Pilbeam (1980) examined photoperiodic schemes
to alter the mink's annual cycle so it so it would be 6
months out of phase with the animals raised in natural
light. They abruptly switched mink on the shortest day
of the natural photoperiod (December 21) to the longest
day. The daylength was then decreased as in nature. The
reproductive performance of these mink was followed for the
next 3 years. The reproductive performance of both the
males and females in the artificial photoperiod was
significantly poorer than the controls on natural light.

12
This could have been due to high ambient temperatures
during the "new" breeding season, endogenous reproductive
rhythms from the original group of mink, and/or improper
light spectra generated by artificial light. These results
proved surprising since two mink ranchers had reported
earlier in a trade journal that they could get some mink to
breed and whelp any time of year (Williams and Turbak,
1970). However, Williams and Turbak's observations were
not as carefully controlled as those to Travis and Pilbeam.
Some mink will respond to artificial photoperiods but not
enough so that it would be feasible for commercial

operations.

3.0) W

3-1) IDLIQQHQELQD

In mammals, the pathway from the ambient photoperiod
through the brain which eventually effects the gonads and
reproductive function, has been partially elucidated. Most
of the anatomical components for mediation of photoperiod
and reproduction have been identified although interaction
has not been fully determined. A simplified model of the
cephalic components of the pathway include: 1) the eye as
the sole photoreceptor, 2) the suprachiasmatic nucleus as
the "time-keeping" portion of the hypothalamus, 3) the
superior cervical ganglia as a regulator of input from the

sympathetic nervous system to the pineal gland, and 4) the

13
pineal gland which converts the neural input concerning
photoperiod into a chemical signal, melatonin (Figure 2).
The pineal will be discussed first and then the other
components of the pathway including how they relate to the

pineal.

3-2) W

The pineal gland is located between the cerebral
hemispheres and anterior to the cerebellum. The pineal
varies in size among mammalian species with a general trend
of smaller glands in animals living near the equator (small
annual changes in photoperiod) and large glands in related
temperate and artic- dwelling species (large circannual
photoperiod changes) (Reiter, 1980a). The discovery of one
of the pineal hormone, melatonin, and its structure revived
interest and research on this gland (Lerner et al, 1958:

1959).

3.2.1) Brief Description of Morphology

Long thought to be a vestigial complex, the pineal
gland arises embryologically from a tubular evagination of
the dorsal diencephalon (Altar, 1982). Pineal parenchyma
(endocrine secretory cells) proliferate rapidly reaching
the greatest rate of mitosis within the first few days
after birth, in the rat. Cell division, mainly of

neuroglia, continues until two months of age. In the rat,

Figure 2.

l4

Presumed components of the photoperiod sensing and
processing system in the mink brain. SCN = Suprachiasmatic
nucleus; PIT = Pituitary gland; SCG = Superior cervical
ganglia; CH = Cerebral hemisphere; CERE = Cerebellum;

. MEL = Melatonin; = Nerve pathway.

___—_ __—___

 

 

 

uum

92.5 452:

 

 

16
neuroglia have a secretory as well as structural role
within the pineal but in the mink they seem to function
only as support structures (Weman and Nobin, 1979). Since
the endocrine cells, or more properly "neuroendocrine
cells", arise from neural tissue they are metabolically
active with many mitochondria and Golgi apparatuses. These
pinealocytes are usually light staining but a few dark
glycogen-rich cells can be found (Rouvet, 1982). They are
surrounded by a thick network of capillaries. Until
recently, it was believed that the only efferent
communication from the pineal was by way of these vascular
connections. However this year, Korf et a1. (1986),

reported the discovery of neural projections from the gland.

3.2.2) Control of Melatonin Synthesis

The pineal gland serves as a transducer of electrical
nerve impulses originating from the retina after photic
input into a chemical message, melatonin. The amino acid
tryptophan is sequestered by the pineal during the day and
is converted in two enzyme-catalyzed steps to the indole
serotonin. (The pineal contains the greatest concentration
of serotonin in the brain.) At the onset of darkness
most of the stored serotonin is converted to melatonin by
another two enzyme system, serotonin n- acetyl transferase
(SNAT) and hydroxyindole-o-methyl-transferase (HIOMT)

(Cardinali, 1981). Since SNAT is the rate-limiting enzyme

17
step, it has been the most studied.

With the onset of darkness, SNAT actvity increases
50-fold and then it falls near dawn (Deguchi and Axelrod,
1972). The circadian changes are endogenous: they are not
driven by the light/dark cycle although they are in phase
with it (Binkley, 1983) and each morning the dawn "resets"
the internal clock mechanism (Ho et al., 1984). If animals
are blinded or in constant darkness, the rhythm persists
for a time but then it "free-runs" (Zatz, 1980).

Although SNAT activity is the primary control of
changes in melatonin concentrations, melatonin synthesis is
also under B- adrenergic control. Synthesis of melatonin
from serotonin is stimulated by B-adrenergic activation or
darkness, and inhibited by B-adrenergic blockage or light
(0xenkrug et al., 1985). It appears that the adrenergic
control point is past the photoperiod control point as
determined by the adrenergic antagonist, naloxone (Sugden
et al., 1985). Naloxone reduces melatonin concentrations in
the blood in the face of darkness-induced increases in SNAT
activity (Lowenstein et al., 1984). Neurons with cell
bodies in the superior cervical ganglia (SCG), synapse on
pinealocytes releasing a B-adrenergic substance,
norepinephrine, in response to the phtoperiod (Bowers et
al., 1984: Coburn, 1984). If the SCG is excised (SCGx),
melatonin is still produced by the pineal but not in phase

with the photoperiod (Deguchi and Axelrod, 1972). SCGx

18

animals lose the ability to measure the length of night and
day and have subsequent disturbances in reproductive
function (sheep: Lincoln, 1979; ferret: Donovan and
VanderWerff ten Bosch, 1956: Herbert, 1967). For example,
ganglionectomy in female mink suppressed the stimulatory
role of long days and the inhibitory role of short days on
post-mating prolactin increases (Martinet et al., 1985).
(Prolactin in mink activates the corpora lutea with
resulting increases in progesterone causing the
termination of embryonic diapause and subsequent
implantation). Afternoon injections of melatonin to pineal-
and SCG- intact bred mink in long days simulated short
photoperiod since secretion of prolactin as reduced and
implantation was delayed (Martinet et al., 1983).

Synthesis of melatonin can also be controlled by
gonadal hormones. The enzyme, HIOMT, is very sensitive to
steriod hormone feedback while SNAT is relatively less so.
HIOMT activity in the pineal paralleled the presence of
melatonin metabolites in rat urine during proestrus (high
estrogen) (Cardinali and Vacas, 1978). However, no such
changes in HIOMT were seen during the estrous cycle of the
hamster, another nocturnal species implying no estrogen
negative feedback mechanism in that species (Rollag et al.,
1979). In cultured male rat pineal gland, both testosterone
and estradiol increase melatonin synthesis (Daya and

Potgieter, 1985). The interrelationship between

19
hormone-related events and the light/dark cycle of activity
of the organ is not yet fully understood but the hormone
effects are generally subsidiary to circadian photoperiodic
forces.

3.2.3) Effects of Melatonin and Pinealectomy on

Reproduction

Early research on the function of the pineal gland
consisted primarily of removing the gland and noting the
subsequent effects. Apparently the investigators expected
immediate results like those seen after hypophysectomy.

The central tenet guiding researchers was that the pineal
and melatonin had negative effects on reproduction, an
"antigonadotropic action." The experiments designed to
test it were inadvertently nonbiological and led to
confusing, inconclusive results (Reiter, 1980b). Reiter
(1973a) summed up their conclusions by stating that
"pinealectomy is a notoriously unreliable means of
illustrating the gonad-inhibiting activity of the pineal
gland” and "in some instances has undetectable effects on
the reproductive system.”

Melatonin sometimes enhanced gonadal development and
prompted some scientists to declare it had "counter-
antigonadotropic" effects (Hoffman, 1974). Reiter (1980b)
offered an explanation for the confusion in a review article
illustrating that the method of melatonin administration,

the time of day it is administered, and the photoperiodic

20
history of the experimental animals all effect the
hormone's action on the reproductive system. Briefly, it
is as follows (the hamster is the model species):

1) Method of Administration: Subcutaneous (SC)
implants of melatonin given to males or females completely
negates the inhibitory effects of short days producing the
counter- antigonadotropic action. It is likely that this
response is abnormal since never in nature is an animal
exposed to the continous presence of melatonin.

2) Time of Administration: Injections of melatonin
given late in the day during stimulatory long photoperiods
cause gonadal regression, whereas those given early in the
day do not. This discrepancy can be explained by the
change in sensitivity in melatonin receptors during the day
(Tamarkin et al., 1976: Chen et al., 1980). After a long
period of melatonin exposure (all night) the receptors are.
"down-regulated” and cannot respond to more hormone.
However, this phenomenon is short-lived, and by late
afternoon the receptors can function again.

3) Photoperiodic History: The length of time the
animals have been exposed to stimulatory or inhibitory
photoperiods, and the inevitability of photorefractiveness,
all determine how they will respond to exogenous melatonin
treatment.

In the mid-1960's it was discovered that the gonads

of hamsters regressed under short photoperiods but that

21

pinealectomy or SCGx blocked the effect thus demonstrating
an antigonadal role for the pineal (Reiter and Hester,
1966). This response has been demonstrated repeatedly by
other researchers (Goldman et al., 1979: Benson and
Matthews, 1980: Bartke et al., 1983). However, in the
closely related Turkish hamster, pinealectomy induces
gonadal regression in long days and does not prevent
regression tin short days demonstrating a progonadal role
for the pineal (Carter et al., 1982). But pinealectomy
prevented any further gonadal responses to daylength
changes in both species. The pinealectomized golden
hamster remains reproductively competent in both long and
short days, whereas the pinealectomized Turkish hamster is
reproductively inactive in either photoperiod (Goldman and
Darrow, 1983). Intact Turkish hamsters must have a certain
length of light (15-17 hours/24 hours) to maintain
testicular function. Daylengths greater than 17 hours or
less than 15 hours induces testes involution (Hong et al.,
1986). The golden hamster needs only photoperiods greater
than 12.5 hours to maintain reproductive function. It is
capable of discriminating between 12 and 13 hours of light
per day.. This intrinsic difference in daylength measuring
ability may account in part for the different responses of
the two species to pinealectomy.

Another long-day breeder, the Djungarian (Siberian)

hamster, has yet another response to pinealectomy. The

22
testes will recrudesce within 9 to 12 weeks after
pinealectomy whether on short or long days, indicating an
antigonadal role for the pineal. However, intact animals
exhibit testicular regrowth after 6 weeks on long days,
suggesting a progonadal role of the pineal (Goldman et al.,
1982).

In the short-tailed weasel, subcutaneous implants of
melatonin for 3 or 4 weeks under long days were sufficient
to stimulate short-day conditions and cause gonadal
regression (Rust and Meyer, 1969).

As discussed above, it is afternoon injections of
melatonin that are the most successful in mimicking short
days and inducing testicular regression (Tamarkin et al.,
1976). Sisk and Turek (1982) demonstrated in hamsters that
melatonin, like exposure to short days, increased the
sensitivity of the hypothalamus and pituitary to negative
feedback by testosterone. They postulated that this was
the means by which melatonin had its antigonadal effects.
If hamsters were actively immunized against melatonin, it
would be expected that they would respond as though they
' were pinealectomized. However, this immunization failed to
negate the antigonadal effects of the pineal in hamsters
exposed to a short photoperiod (Brown et al., 1981).

Anestrous ferrets pinealectomized in the fall did come

into estrous in the spring although several weeks later

23
than controls (Herbert, 1969). It was in the second year
after surgery that the pinealectomized ferrets became
greatly asynchronized from the controls (Herbert, 1972).
When the pinealectomy occurred when animals were estrual,
the surgery did not cause estrus to cease prematurely but in
combination with melatonin injections to stimulate short
days, or short days, estrus was shortened (Thorpe and
Herbert, 1976). It appears that the pineal gland of the
estrus ferret is involved with termination of estrus only
when the animal is exposed to what it perceives as a short
photoperiod.

Some wildlife species that have responded to melatonin
treatments with gonadal regression as they would to short
days include the white-footed mouse (Petterborg and Reiter,
1980), the mountain hare (from Scandinavia) (Kuderling et
al., 1984), and the pallid bat (Beasley et al., 1984).
Melatonin treatments caused wildlife short-day breeders
such as the the white-tailed deer (Bubenik, 1983) and red
deer (Lincoln et al., 1984: Adam et al., 1986) to become
reproductively active. The breeding season of the domestic
sheep, a short-day breeder, can be hastened by exogenous
melatonin treatments (Nett and Niswender, 1982: Arendt et
al., 1983: Lincoln and Ebling, 1985). When pinealectomized
ewes were studied, it was discovered that it_was the

duration of the melatonin infusion, rather than maximum or

24
overall concentration, which was the critical parameter for
inducing reproductive changes (Bittman et al., 1983; Yellon
et al., 1985). Carter and Goldman (1983a) reached similar
conclusions based on their work with the Djungarian

hamster.

3.3) WW
There are a number of parallels in the embryological

development and the biochemistry of the eye and the pineal
gland in mammals. Both are evaginations of the
diencephalon and have some similar cellular structures. In
fact the pineal gland in very young rat pups may be able to
detect photoperiod changes without the benefit of eyes
(Erlich and Apuzzo, 1985). The retina can synthesize
melatonin and has a diurnal rhythm of SNAT (Wiechmann,
1986). However, since pinealectomy is not a natural
occurrence, it seems unlikely that the retina serves as a
major endocrine organ. Small amounts of Melatonin has been
detected in pinealectomized rats and hamsters as well as
those species naturally lacking a pineal, alligator and
armadillo.

The pineal gland contains rhodopsin kinase, an
enzyme previously thought to be restricted to the retina.
The enzyme's activity in the pineal is 68% of the level
found in the eye (Somers and Klein, 1984). Other

biochemical similarities between the eye and the pineal

25
include hydroxyindole-O-methyltransferase (HIOMT) activity
(Cardinali and Rosner, 1971) and the presence of an
S-antigen (Kaslow and Wacker, 1977).

The similarities between the eye and the pineal gland
in mammals are surprising since the major role of the eyes
is very different from that of the pineal. However, both
are part of the pathway which mediates the effects of
photoperiod on reproductive endocrine responses.

Blinded adult hamsters exhibited gonadal regression 9
weeks after surgery although pinealectomized or blinded +
pinealectomized animals did not. However, after 27 weeks
only the gonads of the blinded animals recrudesced (Reiter,
1969). The mechanism for the involution is not fully
understood but is hypothesized to involve a reduction in
prolactin (Prl) and /or luteinizing hormone (LH) release
from the pituitary in the blind animals (Reiter and
Johnson, 1974). Blind and pinealectomized (or superior
cervical ganglionectomized) animals had no reduction in Prl
or LH, but blind but pineal-intact white-footed mice
gonadal atrophy ia associated with increased melatonin and
decreased secretion of LH-releasing hormone (Petterborg and
Paull, 1984).

An interesting observation concerning eye-pineal
interactions with reproduction was made with the golden
hamster mutant, anophthalmic white. These animals are

genetically eyeless and are reproductively infertile.

26
Their gonads are arrested at an immature stage of
gametogenesis. However, after pinealectomy fertility was
restored (Hagan and Asher, 1983). In this case, the
pineal was antigonadal (prevented attainment of full
reproductive competence) even though the lack of ocular
photoreceptors was genetic and present before birth. This
phenomenon is not observed in blind-from-birth humans, but
humans are generally insensitive to photoperiod and require
very bright light to suppress the circadian rise in
melatonin (Lewy and Newsome, 1983).

Within one year after blinding, blinded mink came
into estrus I at the same time as the intact controls
(Thomson, 1954). But later researchers indicated that
asynchrony of reproduction in that blinded or
pinealectomized animals does not develop for at least 2
years after surgery (Herbert et al., 1978). Some
ranch-raised mink experience a progressive retinal
degeneration causing blindness (Hadlow, 1984). This has
potentially serious negative effects on reproduction
causing affected animals to be culled early from the herd.

Pineal-intact, blind ewes continue to follow a
circannual reproductive cycle with or without the presence
of a sighted ram (Legan and Karsch, 1983). Blinded or
pinealectomized cattle exhibit seasonal changes in Prl
secretion independent of temperature (Petitclerc et al.,

1983b). There seems to be an endogenous control of

27
seasonality that is partially independent of photic input

to the eyes or hormonal signals from the pineal.

3-4 W
In a review article, Rappers, Smith and deVries (1974)

discussed the pineal gland's control of hypothalamic
activity. They concluded that "the pineal is not a prime
mover but a regulator of regulators, or an important center
for general homeostasis, very probably exerting its
influence primarily on that most important center of
integration of vegetative as well as of the cerebrospinal
nervous system: the hypothalamus." Since then many of the
details as to how the pineal influences the hypothalamus
have been elucidated.

Receptors for melatonin have been found in the
hypothalamus of rats (Niles et al., 1979). Further
studies have localized the receptive areas to the
suprachiasmatic nucleus (SCN) and the paraventricular
nucleus (PVN) which is on the route from the SCN to the
spinal cord and the superior cervical ganglia. Implants of
physiological amounts of melatonin into the SCN caused
gonadal regression in female white-footed mice within 7
weeks but implants in other areas of the brain had no
effect (Glass and Lynch, 1981).: Lesions to the SCN prevent
male hamsters from responding reproductively to

photoperiodic stimuli (Rusak and Morin, 1976). In fact,

28
pinealectomized rats with SCN lesions did not respond to
daily melatonin injections while pinealectomized animals
with intact SCN did (Cassone et al., 1986) providing
further evidence for the vital interconnection between the
pineal and the SCN. The SCN has sometimes been referred to
as the "endogenous circadian clock" in the control of
reproduction.

Lesions (Pickard and Turek, 1983) and electrical
stimulation (Reuss et al., 1985) of the PVN also block
short-day induced testicular regression in hamsters and
lead to a loss of the nighttime increase in melatonin
(Klein et al., 1983).

Mink in short days for 3 months developed greater and
more active nuclear volumes and secretory structures in the
SCN than mink in long days (Yurisova and Klockov, 1979).

These nucleui, especially the SCN, are critical for
the physiological response to photic input to the eyes and
mediated by the pineal gland. The mechanism and neural
pathways between the SCN, LHRH production, and the
gonadotropins has yet to be determined. There is still a
great deal to learn before the reproductive response to

photoperiod is fully understood.

 

For this discussion the working definition, although
general, will be that of Bronson and Rissman (1986) which
states ". . . puberty is simply the period of accelerated
reproductive development that culminates in functional
fertility, as assessed both physiologically and
behaviorally."

The prepubertal animal has small, infertile gonads,
little sexual interest, and no secondary sexual
characteristics. The testes and ovaries produce very
small amounts of testosterone and estradiol with no gametes
while the pulsatile secretion rate of LHRH and LH from the
brain is very slow although the amplitude of the pulses is
very large (Root, 1973). Usually a pulse of LH is followed
by a pulse of steroids.

As the animal grows older the hypothalamic-pituitary
axis matures and becomes less sensitive to the negative
feedback effects of the gonadal steriods. As the frequency
of gonadotropin pulses increases, the baseline of secretion
rises (more hormone is released overall) but the amplitude
of the pulses is smaller. This process has been termed
"gonadstat resetting" and was first hypothesized by Ramirez
and McCann (1963).

The gonadostat theory of puberty has been studied

intensively in a few species and thus far has not been

29

3O
disproven in describing the neural process. The theory
describes the changes in the hypothalamic-hypophyseal axis
during the onset of the annual breeding season in mature
animals. The changes were first described in detail in the
female lamb (Legan et al., 1977), and later by Ryan and
Foster (1980) then subsequently strongly supported by
studies in the male and female rat (Smith et al., 1977;
Bhanot and Wilkinson, 1983; Bourguignon and Franchimont,
1984: Hatsumoto et al., 1986: Nash et al., 1986; 0jeda et
al., 1986), the hamster (Sisk and Turek, 1983), the pig
(Elasaesser et al., 1978), the heifer (Day at al., 1984),
the male lamb (01ster and Foster, 1986), and the ferret
(Ryan, 1984; Ryan and Robinson, 1985: Sisk, 1986).

The increased secretion of gonadotropin near puberty
stimulates production of steriod only if the gonads are
mature enough to respond (0jeda et al., 1983a). The brain
initiates the sequence of changes leading to puberty, but
the gonads complete this maturational process by responding
and setting up the hypothalamic-pituitary-gonadal
interactions seen in the adult (0jeda et al., 1980).
There does not seem to be a specific physiological
"trigger" for puberty but it is more the result of a
gradual developmental process.

Aside from photoperiod influence, the time of puberty
can be negatively influenced by poor nutrition. Underfed

heifers (Petitclerc et al., 1983a), lambs (Foster and

31
Yellon, 1985), and rats (Holehan and Merry, 1985: Bronson,
1986) will reach puberty later than ;g_119 fed animals even
under a stimulatory photoperiod. However, puberty cannot
be delayed indefinately and will eventually occur even in
very thin females, but then there may be no further
reproductive cyclicity (Bronson and Rissman, 1986). In
males, an energy deficit does not appear to be a major

factor.

4.2)Inmlxemenf__ef_the_rineal_in_the_zh9feperip_dic
II!'!' :21!

As discussed in Part 3.0, the pineal gland is the major
neuroendocrine organ that responds to photoperiod, releasing
melatonin during darkness but not in light. Therefore, in
prepubertal, seasonally breeding mammals, it is the pineal
that "codes" for either increasing daylength (stimulatory
to "long-day breeders") or for decreasing daylength
(stimulatory to "short-day breeders").

Naturally-occurring nocturnal melatonin pulses are
infrequent and of small amplitude in the prepubertal lamb.
However, as the fall breeding season approaches the
frequency and amplitude greatly increase with the largest
amount secreted nocturnally the last part of October
(Rodway et al., 1985). The importance of this melatonin
code in the proper timing of puberty is demonstrated by the

fact that pinealectomized lambs experienced puberty 3

32
months later than intact controls (Kennaway et al., 1985).
Puberty did occur but during the second year the
pinealectomized ewes were out-of-synchrony with the
photoperiod and with the control animals. If lambs are
superior cervical ganglionectomized (SCGx) thereby
denervating the pineal gland at an early age (6 to 8 weeks),
they do not achieve puberty until 65 weeks as compared to
34 weeks for intact controls. Infusing melatonin into SCGx
lambs to stimulate long then short days causes puberty to
occur with that of controls (Yellon and Foster, 1986).

A practical use of melatonin to advance puberty in the
sheep was attempted by Nowak and Rodway (1985). They found
that intravaginal implants in 19-20 week old lambs
initiated puberty 4 weeks before controls. Implants in
younger lambs (7 weeks) did not hasten puberty. This
demonstrates that continuously available melatonin mimicked
short days in lambs with a brain-gonadal axis mature enough
to reproductively respond.

One of the first actions of exogenous melatonin that has
been described was the delay of puberty in rats (Chu et
al., 1964). Rollag et a1. (1982) found that afternoon
melatonin injections in golden hamsters did not prevent
sexual maturity but once it was attained, melatonin caused
rapid gonadal regression. Young Djungarian hamsters raised
in short days showed rapid testicular growth when

transferred to long days. If they were pinealectomized,

33
this growth did not occur without melatonin infusions
simulating long days (Carter and Goldman, 1983a). The time
relative to the onset of light melatonin is administered is
of crucial importance due to circadian changes in melatonin
sensitivity (Rivest et al., 1985).

As with hamsters (Reiter and Hester, 1966),
pinealectomy abolishes the negative effects short days have
on reproduction in the prepubertal white-footed mouse
(Johnston et al., 1982) and they also exhibit a circadian
sensitivity to light and consequently, to melatonin
(Whitsett et al., 1984a). Whitsett and co-workers
(1984a,b,c,d) found that exogenous melatonin delayed
puberty in mice reared in long days. Exogenous melatonin
did not effect age at puberty of mice reared in short days.
Interestingly, exposure of short day and/or
melatonin-treated males to adult females after weaning
hastened sexual development so that puberty was not delayed
in the males.

The clinical use of melatonin to treat disturbances in.
puberty has yet to be attempted in humans but there are
reports of the normal, non-pathological changes in
melatonin concentrations near puberty. Silman et a1. (1979)
attempted to determine the changes in serum melatonin
concentration during the day in prepubertal children and in
those in various stages of pubertal development. They
found that with the onset of pubertal development in boys,

34
there was an abrupt drop in melatonin that remained low
during later developmental stages. No such relationship
was found in girls. Penny (1982) measured overnight
urinary excretion of melatonin in similarily age-grouped
children and found that melatonin metablites increased with
advancing pubertal development but was very low in adults,
both male and female. To further confuse the situation,
Waldhauser et al. (1984) reported that although daytime
serum melatonin concentrations did not change with sexual
maturation, nighttime levels did decrease with development.
One explanation could be that serum levels of melatonin
decrease with age because clearance from the body and
excretion rates are high until adulthood when both serum and
urine levels decline.

Melatonin injected in mid-afternoon caused an increase
in prolactin in pubertal but not prepubertal children.
Decreased growth hormone occurred in the prepubertal group
but no change was detected in the pubertal children. There
was also no change in LH, FSH, or TSH in either group
(Lissoni et al., 1986). There appears to be a change in
the pituitary to melatonin with pubertal development but
not all of the hormones respond in this particular

experimental design.

35
5.0) Seesaw

The future of pineal gland research looks very bright.
No longer should it be ignored as a regulator of
reproductive physiology, especially when manipulations of
the photoperiod are part of the experimental design. The
role of the pineal gland may vary among species and thus
there would be some initial. confusion. However, as more
is learned the more we will understand about the control of

reproduction by photoperiod.

IHIBQDQQIIQH

Hamsters and sheep have been extensively used in
photoperiod and reproduction research. Consequently, a good
deal of information has accummulated about their response
to changes in photoperiod. However, to fully understand
the physiology of photoperiodicity, and the reproductive
response to light in particular, more species need to be
studied in more depth (Bronson and Rissman, 1986). Only
then can an accurate general scheme about mammalian
photoperiodism be developed.

Carnivores are an ecologically important order of
mammals since they usually reside at the top of the food
chain. They are also raised or hunted by humans for
skins, or to provide a service (eg. domestic dogs and
cats). They are, unfortunately, a neglected lot when it
comes to understanding their reproductive- responses to
photoperiod. The European ferret (Mustgla pgtggigg figrg)
is one of the few carnivores which has been studied to any
extent. Limited research on the economically valuable mink
(Mussel; yiggn , has been done but it has centered primarily
on how photoperiod effects the pelt (Martinet et al., 1984:
Rose et al., 1984, 1985) or litter size (Hammond, 1951;

Holcomb et al., 1962; Aulerich et al., 1964: Duby and

36

37
Travis, 1972: Pilbeam et al., 1979). Most of what is known
about mink is in an uncontrolled setting and anecdotal.
There has not been much basic research on the mink using it
as a model species. Therefore, in this study, an attempt
was made to increase what is known about photoperiodicity
and reproduction in mink with special attention to how the
pineal gland is involved. Perhaps later, practical

applications can be made to aid the fur farmer.

9:199:13:
To further understand how information about photoperiod
is processed in the central nervous system of a model

carnivore, the mink.

W:

Alteration of the prepubertal mink's ability to
physically receive or encode photoperiodic signals due to
removal of the eye and/or disruption of the pineal gland
melatonin production pattern results in disturbances in

puberty and the annual reproductive cycle.

W:

The original statistical design was a complete block
with repeated measurement (Gill, 1978) (Table 1). However,
due to death losses among the females, their data was
analyzed using a different design which deleted litter
pairs (Table 2). Thirty-six brown-eyed prepubertal (7-8
months old) pastel mink of each sex from 18 different
litters were divided into six surgical treatment groups
comprised of three litters per treatment. The surgical
treatment groups (twelve animals/group) were either

bilaterally blinded, bilaterally superior cervical

38

39

Table 1: Main statistical design of a double split
plot with sex effect analyzed separately.

WM 511:
Treatment 5
Litter pair/treatment 12
Housing 1
Treatment x housing 5
Litter pair/trt. x housing 12
Bleed 32
Bleed x treatment 160
Litter pair/trt. x bleed .344
Housing x Bleed 32
Bleed x trt. x housing 160

Litter pair/bleed x trt. x housing 424

Total 1187

40

Table 2: Design for analyzing unbalanced data

for females by surgical treatments and housing.

WW
(within housing)

Treatment
Animal/Treatment
Bleed
Bleed x Treatment

Animal/Bleed x Treatment

Total

WM
(within treatment)
Housing
Animal/Housing
Bleed
Housing x Bleed

Animal/Bleed x Housing

Total

if

21
32
32

853

41
ganglionectomized (SCGx), blinded and SCGx, sham SCGx,
blinded and sham SCGx, or intact controls. Then half of
the animals in each surgery group were put in one of two
types of housing: inside in lighting-controlled chambers,
on naturally-occurring photoperiod, or outside in open
sided sheds under commercial mink ranch conditions.

The animals chosen for this study were from litters
containing at least two males and two females to form
littermate pairs of the same sex. All the animals selected
from the same litter were given the same surgical treatment
and then placed inside or outside, one male and one female

assigned to each type of housing.
~ . The specific contrasts were body weight, size of
external genetalia and serum concentrations of steriod
hormones within each sex and type of housing for 1) intact
vs. blinded, 2) SCGx vs. sham SCGx, and 3) SCGx + blinded
vs. sham SCGx + blinded. Specific example: concentrations
of testosterone in the blinded males would be contrasted
with that of the sighted, intact males. Or, the testes
volume of the SCGx males would be contrasted with that of
the sham SCGx males. The assumption was made that the
intact and sham SCGx groups would show the same response as

would the blinded and sham SCGx + blinded groups.

42
W:

The mink were housed individually in 60 x 40 x 40 cm
wire cages with sheet metal sides at the Michigan State
University Experimental Fur Farm. Each cage contained an
attached wooden nest box (30 x 30 x 45 cm). The mink were
fed a conventional (wet) diet composed of ground whole
chicken, commercial mink cereal, ocean fish scrap, beef
tripe, liver, trimmings and lungs (Bleavins and Aulerich,
1981). Enough water was added to give the mixture the
consistancy of hamburger and it was placed on a wire feed
grid on the top of the cages. Unconsumed feed was removed
daily before new feed was given. Food and water were
provided ;g_lipitgm.

The animals housed outside were exposed to ambient
temperatures which in central Michigan (42’ 42' N latitude,
84' 28' longitude) range from -10' C in January to +30° C
in July and to photoperiods of 9 hours/day on December 22
to 15 hours/day on June 21. The animals housed inside were
not exposed to such temperature extremes although the
photoperiod closely approximated the natural light/dark
cycle at an intensity of 160- 220 lux provided by 100 watt

incandescence light bulbs.

WW
All animals received their surgical treatments between

November 15, 1984 and December 20, 1984, the year of their

43
birth. All surgeries were performed under antiseptic
conditions using 70% ethanol as a disinfectant. The skin
in the surgical area was closely shaved and scrubbed with
Betadine surgical scrub. The animals were under
ketamine-xylazine induced anesthesia at a dosage of 100 mg
ketamine (Vetalar, Parke-Davis) containing 2% by volume
xylazine (Rompun, Haver-Lockhart)per kg body weight injected
intramuscularily in the thigh. Although this dosage was
four times greater than that recommended by Moreland and
Glaser (1985), it was found to be necessary in order to
maintain a plane of anesthesia deep enough to permit optical
enucleation. The [average time to initiation of recovery
after surgery was 60 minutes. Although longer than
necessary, it proved beneficial in that it provided enough
time for good clotting and fibrin formation which was then
not easily dislodged during the animal's involuntary
movements during recovery. The animals were allowed a 1 to
7 day recovery period in a warm room where they were
observed 5 to 6 times per day before being returned to their

cages.

MW

1. Bilateral Optical Enucleation.

After preparing the area around the eye (shaving and
scrubbing) of an anesthetized animal careful cuts with a

scapel were made just above the upper and below the lower

44
eyelid to remove the ribbon of skin containing the
eyelashes. Using forceps and then hemostats, the lids were
retracted and the eyeball nearly pulled out of the socket
by its lowermusculature. A loop of 4-0 plain gut suture
was slipped over the eyeball and used to ligate the blood
vessels and nerves behind the orb. Once ligated the
eyeball was cut free just distal to the ligature with a
scapel and removed. A 1 cm square piece of sterile Gelfoam
(Johnson 8 Johnson) was then inserted into the eye socket

to control any bleeding. The eyelids were sutured together
with 4 to 5 stiches of 4-0 PDS suture (monofilament,
absorbable, Ethicon) using an interrupted suture pattern.
Any bleeding that occured was easily controlled by direct
pressure with cotton swabs or gauze pads. The surgical
site was wiped gently with a gauze pad dipped in clean
saline to remove any clotted blood from the area. The
procedure was repeated for the other eye. Two females
developed blocked tear ducts from the surgery and their eye
sockets repeatedly became swollen and sometimes infected.
They were given antibiotics and the fluid behind the eyelid
was expressed. During the last half of the study the
problem did not recur.

2. Bilateral Superior Cervical Ganglionectomy (SCGx).

This ganglia, located lateral to the esophagus between
the manidibles, separates from the cervical sympathetic

trunk of the vagus nerve near the posterior ganglia of the

45
vagus (Field and Taylor, 1969). After the usual surgical
site preparation on an anesthetized animal, an incision was
made lateral and parallel to the esophagus. When the
cutaneous connective tissue and musculature was teased
away, the large white vagus trunk was easily seen. It was
followed anteriorly to the branch point. The SCG is the
more lateral of the two ganglia in that area. It was
lifted carefully out of its bed using glass Pasteur pipettes
so as not to stretch the nerve, then the nerve on either
side of the ganglion was cut and the ganglion removed.
"Little or no bleeding occurred during the surgery. The
skin incision was closed with 4-0 PDS suture using an
interrupted pattern. Any disturbed muscles were not
sutured since they were usually separated by teasing and
had not been cut. The site was gently swabbed with clean
saline and the procedure repeated on the other side.

3. Sham Superior Cervical Ganglionectomy.

This procedure was the same as SCGx except that the
ganglia were only manipulated gently with glass instruments
but were not excised.

4. Combination Surgical Treatments.

Blinding + SCGx and Blinding + Sham SCGx were the
combination treatments. The procedure for each surgery was
as outlined above with the ganglia surgery or manipulation

occurring before the blinding.

46
Cellestien_ef_fileed

Blood samples were taken by jugular venipuncture every
two weeks beginning just before surgery and continuing
through the second breeding season in 1986. The animals
were lightly' anesthetized with an intramuscular injection
of the ketamine-xylazine mixture described above at a
standard dosage of 0.2 ml for the males and 0.1 ml for
females which approximated 10 mg ketamine/kg body weight.
This dosage was derived by trial and error and represented
a compromise between the level of sedation needed for blood
collection and a rapid recovery from anesthesia. The fur in
the ventral area of the neck over one jugular vein was
shaved to the skin and swabbed with 70% ethanol. Three
milliliters of blood were drawn from the jugular vein using
a 3 cc syringe and a 20 gauge 1" needle. The blood from
each animal was allowed to clot in individually labeled 12
x 75 mm test tubes at room temperature then refrigerated at
4’C overnight. The serum was collected after
centrifugation at 1800 x g for 20 minutes the next morning.
The serum was frozen at -20‘ C until assayed for
testosterone (males) or estradiol (females).

During the last two-thirds of the study a few animals,
independent of treatment, began to exhibit seizures during
anesthesia. The seizures were much more violent than the
usual resistance to anesthesia, of longer duration, and

became worse during subsequent blood collections. Two

47
animals never recovered and died while under anesthesia. A
third animal that failed to recover from anesthesia did not

exhibit any unusual seizure activity.

Went:

After each blood collection several physical
measurements were taken. The scrotums of the male mink
were palpated, and if testes were located, their length
(L), width (W), and thickness (T) were measured with
calipers through the scrotal wall. A mean testes volume
(TV) was calculated for each animal using a modified
equation for a solid elipse (Boisson-Agasse et al.,1982):

TVa 4/3 x L/2 x W/2 x T/2

‘The vulvae of the females were examined to determine
the amount of estrual swelling or edema. The degree of
edema was scored on a scale of 0-3 with zero representing
the smallest size and three the largest as described by
Travis et al.(1978). The size of testeS‘ and the extent of
vulvar edema were used as external indicators of the level
of sexual competence.

Body weights were measured to the nearest 5 g and an
evaluation of the stage of pelage maturation was made. The
condition of the pelage/coat was scored on a scale of 1-5,
roughly following the molting pattern outlined by Travis and
Schaible (1960). A score of 1 represented animals with

extremely mottled, patchy coats with many loose hairs. A

48
score of 5 was assigned to animals with uniform, fully,
prime coats with well attached hairs. The scores were not
grading scores indicating the quality of the pelt but were
used to indicate the condition, or degree of molting, of the

coat.

WM

Male and female animals were paired during their first

and second breeding seasons to determine if sexual interest
paralleled the physical changes in external genitalia.

The frequency of pairing followed standard mink farm
practice. The animals were placed together the first of
March and if the female was not receptive then she was
tested again four days later. When a successful mating as
determined by "sperm-checking" was obtained, the female was
provided an opportunity for a second mating every four days
until the end of the breeding season (March 24-30), or
until she was willing to accept the male.

The first year the paired males and females were
observed for sexual behavior but they were not permitted to
fully copulate since it was thought that a pregnancy and
lactation might serve as an endogenous "clock-setter",
confounding treatment effects in the female. The pairings
were random with regard to treatment but were kept within
type of housing. The pair was kept together for at least

20-30 minutes while the observer remained out of sight but

49
checked on the pairs periodically. The second year all the
animals that would mate were allowed to do so. The success
of the mating was determined by the presence of sperm in
post-coital vaginal smears, "sperm-checking" (Shump et al.,
1976).

Males were deemed sexually interested if they grasped
the female by the back of the neck while emitting a low
"chuckling" sound always given during courtship (Linscombe
et al., 1982) and attempted mounting and clasping with the
front legs. Females were regarded as sexually receptive if
they did not vigorously resist the—male or attack him and

permitted the grasping and mounting.

MW

1. Testosterone.

Concentrations of testosterone in serum from males were
quantified by the method used by Louis et al. (1973) for
progesterone but with modifications as described by Kiser et
al. (1978) and following: MSU #74 antiserum to
testosterone was diluted 1:10,000 and 3H-testosterone was
used at 5000 cpm/200 ul. Only 50 ul of mink serum were
extracted and assayed instead of the usual 200 ul.

2. Estradiol.
Concentrations of estradiol (E2) were quantified with
modifications of an assay described by England et al.

(1974). Modifications included extracting 200 ul serum in

50
2 ml anhydrous ethyl ether and vortexing vigorously for 2
minutes. After addition of Ez-antiserum (1:200,000 in
phosphate buffer with EDTA and 1:400 normal rabbit serum),
a 2 hour room temperature and 1/2 hour 4' C incubation,
1251-E2 in buffer was added to all tubes and the tubes
vortexed and incubated 1 hour at 4° C. The bound and free
125I-E2 was separated by the addition of 1 ml cold
Dextran-coated charcoal to each tube (2.5 g washed neutral
Norit charcoal with 0.25 g Dextran T70 in 1000 ml phosphate
buffer). This was followed by a 15 minute incubation at
4° C, and centrifugation at 2,500 x g for 15 minutes at
4° C. The supernatant was decanted and counted in a gamma

counter.

RESELIS

In almost all categories, the responses of the animals
to the treatments were so distinct that they could be
combined into two groups: sighted and blinded, despite
other treatment combinations. The sighted group consisted
of superior cervical ganglionectomized (SCGs), sham SCGx,
and intact animals. The blind group was comprised of
blinded, blinded + SCGx, and blinded + sham SCGx animals.
Unless otherwise stated, the results will be pooled and

discussed in terms of these two categories.

Might

Sighted males and females showed circannual
fluctuations in body weight independent of housing (Figures
3 & 5). The SCGx females were significantly (p< .05)
heavier after the first breeding season than the other
sighted females. Most of the increase was due to the
indoor-housed animals in that group. All of the blinded
males had a significant (p< .05) reduction in body weight
after the first breeding season which then did not increase
throughout the rest of the study (Figure 4). The blinded
females showed more circannual changes in body weight,
although not significant, than the blind males but not as

much as the sighted females (Figure 6).

51

52

Figure 3. Changes in body weight over time in sighted male
mink across housing type (n = 6/treatment).

53

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54

Figure 4. Changes in body weight over time in blinded male
mink across housing (n = 6/treatment).

55

 

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56

Figure 5. Changes in body weight over time in~sighted
female mink across housing (n = 7 intact,
4 sham SCGx, 6 SCGx).

 

 

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58

Figure 6. Changes in body weight over time in blinded
female mink across housing (n = 4 blind,
5 sham SCGx + blind, 5 SCGx + blind).

59

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60

WW

There was an interaction (p< .001) between time of
bleed and surgery as well as among surgery groups for
testes volume. Testes size in sighted males was smallest
in June while the blinded males did not achieve minimal
size until September (Figures 7 & 8). The SCGx males began
testes recrudesence in November, significantly earlier
(p< .05) than other sighted males that did not start to
show regrowth until the end of December. Recrudesence did
not occur in blinded males.

'There was an interaction (p< .001) between time of
bleed and surgery as well as among surgical groups for
vulvar edema. The blind females maintained enlarged vulvae
longer than sighted animals and did not show an estrual
swelling the second year (Figures 9 & 10). The vulvar
enlargement of the sighted animals in the second year was

less than the first.

W

Testosterone (T) concentrations paralleled and preceded
the changes in testes size in sighted and blinded males.
There was no effect of housing on T levels but there was
an interaction (p< .001) between time of bleed and surgery
as well as among surgery groups. The blind males had

higher T levels for a longer period of time than the

61

Figure 7. Changes in testicular volume over time in sighted
male mink across housing (n = 6/treatment).

62

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63

Figure 8. Changes in testicular volume over time in blinded
male mink across housing (n = 6/treatment).

64

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65

Figure 9. Changes in vulvar edema over time in sighted
female mink across housing (n = 7 intact,
6 sham SCGx, 6 SCGx).

66

 

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67

Figure 10. Changes in vulvar edema over time in blinded
female mink across housing (n = 4 blind,
S sham SCGx +:blind, 5 SCGx + blind).

68

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69
sighted males the first year and then showed no increase in
T concentrations the second breeding season (Figures 11 &
12).

Estradiol (E2) concentrations did not differ between
sighted and blinded groups of females. There was no
parallel of E2 with vulvar swelling except perhaps the
second breeding season in which E2 titers were higher in

sighted animals than in blinded animals (Figures 13 & 14).

HaiI_QQi§

The pelage was not evaluated until about one-third of
the way through the study (June 1985). It was noted that
the blind animals were not shedding their winter coats and
growing summer ones. Blind males and females had coats
that were in much poorer condition than the sighted animals
throughout the study after the first breeding season
(p< .001). The coats improved in September and October but
they never "primed." The sighted group molted normally and
their coats were prime in December during the usual

pelting time.

;Bepzsxnuztiystjganrzigr:

The first year essentially all the animals showed
sexual interest but were not allowed to mate completely.
. The second year only the sighted males mated successfully

with sighted females. Blind males showed some sexual

70

Figure 11. Changes in testosterone concentrations over time
in sighted male mink across housing (n = 6/treatment).

71

TEstosterone (ng/ml)

 

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72

Figure 12. Changes in testosterone concentrations over time
in blinded male mink across housing (n = 6/treatment).

73

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74

Figure 13. Changes in estradiol concentrations over time
in sighted female mink across housing (n = 7 intact,
4 sham SCGx, 6 SCGx).

75

 

 

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76

Figure 14. Changes in estradiol concentrations over time
in blinded female mink across housing (n = 4 blind,
S sham SCGx + blind, 5 SCGx + blind).

77

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78
interest even though their testes were very small but the
interest was more aggressive in nature. The blind females
had some interest in the males the first year and none the
second year.

The sighted males housed outdoors had more successful
matings as verified by vaginal post-coital sperm checks than
those housed indoors. The group that had the largest
number of matings was the SCGx males with a total of 21.
The intact males were next with 6. The sham SCGx males had
no verified good matings.

All the sighted females were mated, and of them 66%
whelped. Those not producing litters were evenly
distributed across the three sighted groups. The average
litter size at birth was 5.80 for intact females, 3.25 for
SCGx, and 2.66 for sham SCGx. There were too few females

in each group to determine any statistical significance.

DIEQESEIQH
Man:

The cyclical pattern of weight gain and loss in the
sighted mink closely parallel the annual changes in
daylength. As the days lengthen the animals lose weight
which they then gain as the days shorten. Ambient
temperature also follows an yearly cycle, increasing when
days lengthen and decreasing as they shorten. However, a
role of temperature changes in circannual weight changes
can be discounted since: 1) blinded animals showed no such
pattern, although the females showed a trend in that
direction, and they were exposed to the same temperature
conditions, and 2) the body weight changes also occurred in
mink housed indoors, sheltered from the stress of large
shifts in temperature. The importance of photoperiod over
ambient temperature has been reported in cattle (Petitclerc
et al., 1983a), hamsters (Steinlechner et al., 1983;
Bartness and Wade, 1984; Vitale et al., 1985) and mice
(Hutchinson and Hart, 1984: Andrews and Belknap, 1985).

It is not clear why the indoor SCGx females were so
much heavier than the other sighted female groups. It was
not due to genetics pgz_§§, since their brothers in the
male SCGx group were not heavier than other sighted males.
It is possible that the combination of the surgery and

housing synergized within a naturally-occurring female

79

80
annual cyclicity to cause those animals to gain more

weight.

W

Clear evidence of the importance of photoperiod in
controlling the annual breeding season is demonstrated by
the changes in testes size of the sighted and blinded
males. Not only does photoperiod control the onset of
testes growth but also appears to control its regression
after the breeding season since the blinded animals had a
slower rate of testes decline than sighted males. The
decline in testes size seems inevitable and the lengthening
photoperiod only hastens it. However, recrudesence needs
to be stimulated by decreasing daylength (Onstad, 1967).
Those animals unable to visually detect light do not
spontaneously prepare for the breeding season.

The SCG may have an inhibitory or modulatory role in
testes regrowth,since the SCGx males began testes
enlargement over one month sooner than the other
ganglia-intact animals. This effect has not been described
previously, although SCGx animals do maintain large testes
longer (Lincoln, 1979).

The pattern of vulvular size changes in the females is
very similar to the testes size changes of the males. The
blind females did not exhibit estrual swelling at all the

second year. The sighted females showed swelling both

81

years but not as much the second year. This could be due to
the repeated stress of handling, anesthesia, and blood
collection, or it could be the result of copulation which
was permitted that season. The swelling is positively
correlated with blood estradiol concentrations (Travis et
al., 1978) and since mink are induced ovulators, mating
would cause ovulation and reducted synthesis of estradiol.

Based on genital changes both sexes achieved puberty
whether sighted or blinded. They could have very well
gotten their critical photoperiodic cues for puberty before
the surgical treatment. It is very difficult to prevent
puberty totally. It may occur but then the animal become
secondarily infertile. This has been noted in heifers on
restricted intake diets (Petitclerc et al., 1983a), lambs
(Foster and Yellon, 1985), and rats (Holehan and Merry,
1985; Bronson, 1986). It can also be delayed but not
prevented by certain social interactions in mice (Coppola
and Vandenbergh, 1985) and voles (Rissman and Johnston,
1986), and by alterations of central nervous system
activity (Reiter and Ellison, 1970; 0jeda et al., 1983b;

Rivest et al., 1985).

, MW
There was a very close relationship of testosterone (T)
concentrations in serum and testicular size independent of

surgical treatment. It is interesting that changes in the

82
T levels preceded measurable changes in testes volume.
This could be explained by the fact that testosterone is
needed to stimulate the whole spermatogenic apparatus and
thus there would be a lag between the time T titers begin
to rise and the semineferous tubules enlarge (Onstad, 1967;
Sundquist et al., 1984). Also the testosterone
radioimmunoassay is more sensitive to changes than are
calipers. ‘

Although the females' genitals changed with photoperiod
and proximity to the breeding seasons, serum estradiol
(3;) did no follow the external changes in an obvious
manner. At first there appeared to be no difference in
concentrations of 32 between the blinded and sighted
females although the sighted group seemed to have a higher
baseline and mean level the first 10 months (approximately
70 vs. 55 pg/ml). Both groups showed a nadir from November
to January then increase in February and March. The
sighted females showed a greater increase in concentrations
of E2 than the blind ones. Why there is higher E2 during
the summer and fall is unknown. This observation has not
been reported before. The radioimmunoassay for 32 had an
interassay variation of 42%. Consequently the estradiol
concentrations described here may not be as reliable as

would be desired.

83
W

Examination and evaluation of pelage was not part of
the original design and was only begun because it was noted
that the blind animals were not shedding their winter coats
in June like the controls. The appraisal of the coat
condition was subjective but was based on the experience of
two knowledgable mink researchers. However, the results
show definative differences between the sighted and blinded
groups.

Hair coat quality is not a direct measure of
reproduction, but it is further evidence for the importance
of optic photoreceptors as daylength detectors in the
central processing of photoperiodic information. The blind
animals' coats were "normal", in phase with control
animals, until after puberty and then they began to molt in
abnormal patterns and incompletely. Usually the molt or
priming of the fur begins with the tail and proceeds
anteriorily with the head molting or priming last. The
blind mink had mottled, blotchy hair growth with some

patches which never shed.

WW

Behavior was used to determine if the changes in
genital sizes were true indicators of sexual competence
which includes the desire to mate or to be mated. The

enlarged testes of all the males the first year were

84
accompanied by sexual interest but not the second year.
Surprisingly, even the blinded males whose testes were
small and abdominal went through some of the copulatory
behaviors such as "chuckling", grasping the back of the
female's neck, dragging her around the cage, attempting to
position her for coitus (MacLennon and Bailey, 1972). They
did not successfully mate, though, and usually ended up in
a serious fight with the female.

The sighted males housed indoors were not as vigorous
breeders as those kept outside. Only one indoor male had a
successful mating. A successful mating was determined post-
coitally by vaginal swabbing for sperm checked under a
microscope (Sundquist and Gustafssen, 1983). The SCGx
males had the greatest number of good matings, the intact
males had less, and the sham SCGx males had none. This
latter group contained two mink from one litter that had
only one testis descended and this might have had a
negative effect on their breeding performance (Hartung and
Seffner, 1983). The SCGx group had testes enlargement
significantly earlier than the other groups and it was
suspected that they may be out of reproductive condition
during the normal breeding season. This was not the case as
they were the most vigorous breeders.

The reproductive performance of females was actually
better than for males. The first year all the females

would have permitted mating, while the second year only the

85
sighted females mated. All females within the sighted
group were mated at least once during the second season and
66% of them whelped. This is lower than average
reproduction for a commercial mink ranch but these animals
had not been mated the previous year and it can be surmised
that this might have had a detrimental effect on their
reproductive performance. Numerically, intact females had
the largest average litter size, there were too few litters

to make any valid conclusions.

In this study it is the eyes that are the most crucial
component in the eye-SCG pathway to the pineal in mink since
without them the animals became desynchronous with the
photoperiod despite other treatments. There was no
spontaneous regeneration of the gonads and the
post-breeding season atrophy of the testes was slowed in
blinded animals indicating that mink require the
photoperiod to stimulate reproductive function as well as
to hasten its end.

The SCG may have an inhibitory role in gonadal
recrudesence since without it the testes of the male became
larger sooner than in intact males. However, the effect of
the eyes overrode that of the ganglia since blind + SCGx
animals responded the same as blinded-only animals. The
SCGx females responded the same as other sighted females so
the role of the ganglia in sighted mink is not well
defined.

86

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