Title: Studies of general and sexual development in voles (Microtus)
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00099563/00001
 Material Information
Title: Studies of general and sexual development in voles (Microtus)
Physical Description: viii, 234 leaves : ill. ; 29 cm.
Language: English
Creator: Salo, Allen L., 1963-
Copyright Date: 1992
Subject: Voles -- Development   ( lcsh )
Voles -- Reproduction   ( lcsh )
Psychology thesis Ph. D
Dissertations, Academic -- Psychology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Statement of Responsibility: by Allen L. Salo.
Thesis: Thesis (Ph. D.)--University of Florida, 1992.
Bibliography: Includes bibliographical references (leaves 195-209).
General Note: Typescript.
General Note: Vita.
 Record Information
Bibliographic ID: UF00099563
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 001867926
oclc - 28997619
notis - AJU2442


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This work is dedicated in remembrance of my father,

Raymond John Salo, who conducted comparative research on

two species of rabbits at the University of Massachusetts.


I would like to sincerely thank a number of people who

helped me complete the Ph.D. requirements. Although I am

not able to list all individuals who helped me in this

regard, a few should be named. First, I would like to thank

members of my family who supported my sometimes arduous trek

through academe. Most importantly my mother, Sonja Salo,

supported me spiritually as well as financially when the

going became difficult. My brother Mark Salo, his wife

Laurie, and children, were also constant sources of

inspiration. Other significant individuals include past and

present fellow graduate students: Florence Caputo, Jo

Manning, John Pierce, Joan Scheffer, Steve Taylor, Betty

Inglett, Sue Halsell, Nick Mills, and Kerry Sheehan.

Faculty members who have most strongly supported my

endeavors include Dr. Donald Dewsbury, Dr. H. Jane

Brockmann, and Dr. Jeffrey French. Certainly many other

members have impacted my life in various, positive ways.

Finally, I would like to thank members of the staff at the

University of Florida who helped in one way or another;

these individuals include: Theodore Fryer, Isaiah

Washington, Chris Wilcox, and Jeanene Griffin.



ACKNOWLEDGEMENTS... ................................... 11iii

ABSTRACT..... ......... ................................. vii


1 GENERAL INTRODUCTION ........................ ...... 1

Statement of the Problem ......................... 1
Plan of the Dissertation......................... 4
Definitional Considerations...................... 5
A Review of Mating Systems ....................... 6
Puberty Modulation in House Mice
(Mus musculus)................................. 7
The Social Biology of Voles (Microtus)............ 16
Puberty Modulation in Voles (Microtus)........... 19
Principles Underlying Puberty Modulation
in House Mice and Voles........................ 27
Problems with Previous Studies of Puberty
Modulation in Microtus......................... 38

APPARATUS...................................... 41

(EXPERIMENT 1).................................. 43

Rationale. ....................................... 43
Method. .......................................... 45
Subjects. ...................................... 45
Procedure ......................................... 46
Statistical Analysis........................... 49
Results .......................................... 50
Body Weight .................................... 51
Anogenital Distance............................ 51
Adrenal Weight................................. 52
Testes Weight.................................. 54
Seminal Vesicle Weight........................ 54
Ovarian Weight. ............................... 54
Uterine Weight. ............................... 54
Status of Vaginal Perforation................. 56
Delay until Vaginal Perforation............... 57
Vaginal Smears. ............................... 58

Discussion .............................. ...... 65
Body Weight..................................... 65
Anogenital Distance............................ 67
Adrenal Weight................................. 69
Testes Weight.................................. 70
Seminal Vesicle Weight.................. ...... 71
Ovarian Weight................................. 72
Uterine Weight. ................................ 73
Characteristics of Vaginal Perforation........ 75
Vaginal Cytology............................... 77
Conclusions (Experiment 1)...................... 80

(EXPERIMENT 2).................................. 116

Rationale........................................... 116
Method.............................................. 121
Subjects...................................... 121
Procedure...................................... 121
Statistical Analysis........................... 123
Results ......................................... 124
Body Weights. ................................. 229
Vaginal Smears................................. 231
Preference Tests.............................. 124
Differences in the Time Near the Stimuli
Across Weeks.................................. 126
Between-Species Comparisons................... 127
Duration within Center of Cage:
Within-Species Analyses..................... 130
Duration within Center of Cage:
Between-Species Analyses .................... 130
Discussion.......................................... 132
Olfactory Preference Tests .................... 132
Between-Species Comparisons................... 134
Duration within Center of Cage................ 136
Conclusions (Experiment 2)...................... 137


Rationale........................................... 145
Method.............................................. 151
Subjects. ..................................... 151
Procedure...................................... 151
Statistical Analysis.......................... 152
Results ......................................... 153
Delay to Produce Litters...................... 153
Number of Offspring Born....................... 155
Age 50% of Offspring Opened Eyes............... 156
Number of Offspring Weaned.................... 157
Sex Ratio of Offspring Weaned................. 157
Body Weight of Offspring Weaned............... 158

Discussion. ..................................... 161
Delay to Produce Litters....................... 161
Number of Offspring Born....................... 163
Age 50% of Offspring Opened Eyes.............. 164
Number of Offspring Weaned.................... 165
Sex Ratio of Offspring Weaned................. 166
Body Weight of Offspring Weaned............... 167
A Reanalysis of the Evolution and
Expression of Male Parental Behavior......... 169


Overview of Experimental Results................ 183
An Attempt at a Synthesis........................ 183
Routes of Future Study........................... 190

REFERENCES............................................ 195




MICROTUS (EXPERIMENT 1)....................... 218


MICROTUS (EXPERIMENT 2)....................... 231

BIOGRAPHICAL SKETCH................................... 234

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy



Allen L. Salo

December, 1992

Chairman: Donald A. Dewsbury
Major Department: Psychology

Three experiments were conducted to investigate how

patterns of general and sexual development might be

correlated with the formation of social and mating systems

among four species of voles (Microtus). Species included

pine voles (Microtus pinetorum), prairie voles

(M. ochrogaster), meadow voles (M. pennsvlvanicus), and

montane voles (M. montanus). In Experiment 1, general and

sexual development were monitored as voles were exposed to

pheromones contained in the soiled bedding from family

groups, adult males, or adult females. Few significant

effects were found to be due to the treatment. Male pine

voles exposed to family or male bedding were significantly

heavier than those exposed to clean or female bedding. The

uteri of female montane voles exposed to clean or male

bedding were heavier than the uteri of those exposed to

family or female bedding.

In Experiment 2, the olfactory preferences of voles

were measured when they were exposed to male and female

bedding on weeks 4, 7, and 10 after birth. Few preferences

were shown for either bedding type by any of the species.

Female prairie voles and meadow voles revealed a significant

preference for male versus female bedding. Both sexes of

all species differed little in the total duration they

remained near the female stimulus. Male meadow voles

remained near the female stimulus significantly less than

the males of the other species on week 10. Female montane

voles remained near the male stimulus significantly longer

on weeks 4 and 7 than did females of the other species.

In Experiment 3, the influence of the fathers' presence

and absence was studied during the rearing of the breeding

pairs' first two litters. Pine voles produced their second

litter considerably earlier if the male had been present

during the rearing of the first litter rather than being

absent. Pine voles weaned heavier offspring in the second

litter than in the first, when the male had been present for

the rearing of the first litter. Montane voles produced

litters that were male-biased in sex ratio across both

litters, if the male had been present during the rearing of

the first litter.

Results are discussed and interpreted from known

differences in their contrasting social and mating systems.



Statement of the Problem

Several theories have been advanced within the last two

decades to account for the evolution of different social and

mating systems among birds and mammals (e.g., Emlen & Oring,

1977; Kleiman, 1977; Orians, 1969; Vehrencamp & Bradbury,

1984; Wittenberger 1979). However, not all of the selective

forces that underlie the evolution of different mating

systems have been identified (see Vehrencamp & Bradbury,

1984). Wittenberger (1979) proposed that because mating

behavior is affected by nearly all aspects of an organism's

behavioral adjustment to its environment, a theory of mating

systems must be integrated with several other types of

behavioral theories. For example, theoretical advances

concerning the evolution of territoriality, parental

behavior, and sociality must be meshed within a broader

theory explaining the evolution of mating systems.

One area of study that appears promising for providing

greater insights into the evolution of social and mating

systems in mammals is research on the regulation of sexual

maturation or puberty modulation. For example, although its

function remains obscure, delayed sexual maturation is often

present among mammalian species that are considered to be

monogamous (Kleiman, 1977; Dewsbury, 1981). Some have

suggested that delayed sexual maturation functions to

inhibit incestuous mating (McGuire & Getz, 1981), although

others have suggested it reduces susceptibility to predation

(Batzli et al. 1977). Investigating the causes and function

of changes in sexual maturation may lead to an increased

understanding of the evolution of different social and

mating systems.

A second area of investigation that may offer insight

into the formation of the different mating systems is the

evolution of male parental care. Paternal care is found

most often among mammalian species that form monogamous

mating systems and have relatively few offspring (Kleiman,

1977; Dewsbury, 1981). Although it is often assumed that

paternal behavior has a beneficial effect on the development

of young, the assumption needs to be validated (Wuensch,

1985). The results of several studies conducted to

investigate this hypothesis among species of rodents are

equivocal (Dewsbury, 1988). It is possible that there are

important links between sexual suppression and the evolution

of paternal behavior, because the two often occur jointly in

monogamous mating systems (Kleiman, 1977; Dewsbury, 1981).

Until recently, the study of developmental processes

has been largely divorced from studies of the evolution of

mating systems. Muller (1990) noted that ontogeny has been

treated as a type of "black box" in evolutionary theory.

Similarly, Stearns (1989) discussing developmental

processes, noted that the types and sources of phenotypic

variation have been given little consideration in

evolutionary theory. The following set of experiments was

designed to investigate possible causal links between

puberty modulation and paternal care and the resulting

differences in social and mating systems in mammals.

Species of the genus Microtus are ideal for studying

the functions of sexual maturation and paternal behavior for

three primary reasons. First, the species display

differences in social and mating system that appear to be

associated with puberty modulation and paternal care. For

example, the mating systems among Microtus range from

monogamy in prairie voles (M. ochrocaster) to promiscuity in

meadow voles (M. pennsylvanicus)(Wolff, 1985). Pine voles

(M. pinetorum) and prairie voles are considered to be

monogamous and appear to be sensitive to the actions of

pheromonal cues that affect sexual maturation (Getz &

Hofmann, 1986; FitzGerald & Madison, 1983; Carter & Getz,


A second reason for studying these processes in

Microtus is that by investigating differences in puberty

modulation and paternal behavior with closely related

species, we are most likely to identify the selective

pressures and mechanisms that have shaped these species

differences (Clutton-Brock & Harvey, 1984; King, 1970;

Dewsbury, 1990). A third reason is that many species of

Microtus can be bred and maintained within the laboratory

where it is possible to systematically vary and control

exposure to stimuli. Such identification and control of

stimuli is difficult or impossible with these species under

natural conditions. The control offered in the laboratory

appears to be a prerequisite to identify the necessary and

sufficient stimuli that influence sexual maturation among

species. Finally, although there is a considerable

literature on such topics as the taxonomy, zoogeography,

anatomy, and habitats, of various species of Microtus,

currently little is known about many developmental processes

such as prenatal development, causes of interspecific

variation in litter size, and causes of mortality (Tamarin,

1985; Nadeau, 1985). It is possible that through the

systematic investigation of such phenomena, relationships

will become evident between them and the selective pressures

within the environment which shaped them.

Plan of the Dissertation

Three studies are presented that were designed to

investigate the role of two developmental phenomena that

might have influenced the evolution and expression of the

different social and mating systems among four species of

voles (Microtus). The two developmental phenomena include

the changes in the timing of puberty (puberty modulation)

and influence of paternal presence or absence upon

developing offspring. Specifically, Experiment 1 was

designed to explore how exposure to naturally occurring

pheromones, contained within soiled bedding, influences the

timing of puberty in four species of voles. The species

included pine voles (M. pinetorum), prairie voles,

(M. ochrogaster), meadow voles (M. pennsylvanicus), and

montane voles (M. montanus). In Experiment 2, the

behavioral reactions of the four species were studied when

they were exposed to soiled bedding in an odor preference

task. The study was designed to determine if behavioral

preferences for male and female soiled bedding were evident

within each species and sex and whether the preferences

changed as a function of age. In Experiment 3, the effect

of an adult male's presence or absence on his developing

offspring was studied. Together the three experiments were

designed to explore how these phenomena were related to

development and how they might be functionally linked to the

expression of different social and mating systems.

Below I present some definitional considerations,

present a review of the literature on puberty modulation in

house mice (Mus musculus) and voles (Microtus), present a

review of the social biology of Microtus, and present the

results of the studies designed to investigate puberty

modulation and the effects of paternal presence in Microtus.

Finally, I attempt to synthesize the results of the three

studies with results from other studies to explain possible

selective pressures that might have formed and may maintain

the different social and mating systems among these species.

Definitional Considerations

There is no globally accepted definition of puberty.

According to Hasler (1975), puberty has usually been defined

as the age that animals produce viable gametes. However, in

practice, it is often difficult to define the limits of

puberty with precision (Bronson & Rissman, 1986). Common

measures used in studies with female house mice have

included the day of vaginal opening or the first day of an

estrous vaginal smear (Drickamer, 1986). It has proven

difficult to find a reliable and non-terminal marker to

assess puberty in male mice. The presence of viable

spermatozoa or the ability to induce pregnancy have been

used in some studies as an index of puberty among males

(e.g., Vandenbergh, 1971).

Despite debate over how one defines puberty or what one

uses as an index of puberty, by choosing a standard measure

it is possible to compare differences in the onset of

"puberty" as being either accelerated or delayed compared

with some reference group. Thus, the term "puberty

modulation" is useful when referring to the onset of puberty

as being either accelerated, delayed, or both, when

comparing individuals or groups.

A Review of Mating Systems

The mating system of a population can be regarded as

the ensemble of behaviors and physical adaptations specific

for mating that are available to a population (Vehrencamp &

Bradbury, 1984). The mating system of a population is an

emergent property that reflects the traits and propensities

of the individuals within the population (West-Eberhard,

1979). For example, characteristics of a monogamous mating

system among mammals include (1) the continual close

proximity of an adult heterosexual pair during and outside

periods of reproduction, (2) mating preferences, (3) the

absence of unrelated adult conspecifics from the breeding

pairs home range, and (4) breeding by only one adult pair in

a family group (Kleiman, 1977). Other common forms of

mating systems include polygyny, in which there is a

prolonged association and essentially exclusive mating

relationship between one male and two or more females at one

time, and promiscuity, in which there is no prolonged

association between the sexes and multiple matings by

members of at least one sex (Wittenberger, 1979). The goal

of research has been to identify the primary selective

pressures or foci that have created the behavior differences

associated with each mating system, with the use of

appropriate theory and field work (Vehrencamp & Bradbury,


Puberty Modulation in House Mice (Mus musculus)

Most investigations of the phenomenon of puberty

modulation have been conducted with house mice

(Mus musculus). Researchers have identified several factors

that can influence the timing of puberty in young female

mice. Many studies have used indices such as the day of

vaginal perforation or the detection of an estrous smear as

a reliable means of assessing puberty. Considerably less

research has been designed to investigate the factors that

affect puberty in male mice. However, research with both

sexes appears necessary to understand how differences in the

timing of puberty might affect the expression of social and

mating systems.

A number of factors have been shown to influence the

timing of puberty in female house mice. Broadly, these

factors have included genetic differences (Drickamer, 1981),

social conditions (see Drickamer, 1986 or Vandenbergh &

Coppola, 1986 for recent reviews), and non-social factors

such as temperature (Barnett & Coleman, 1959), photoperiod

(Drickamer, 1975a), and season (Kruczek & Gruca, 1990). The

current review and experiments have been based on the

investigation of social factors that have been shown to

influence puberty. Specifically, chemosignals or

pheromones, found in the urine of several muroid species,

have been shown to differentially accelerate or retard the

attainment of puberty in young individuals (Levin &

Johnston, 1986).

At least four types of urinary chemosignals can

influence puberty in female mice (Drickamer, 1986). These

signals include urine from (1) male mice, (2) pregnant or

lactating females, (3) females in estrus, and (4)

group-caged females. The first three signals accelerate the

timing of puberty relative to female mice housed similarly

but not exposed to the chemosignals. The last signal delays

puberty in females compared to those not exposed to the

signals. Below, I present the principal findings associated

with each of the four stimuli that affect puberty in female

house mice.

Puberty Acceleration Caused by Male Urine

Not all urine from male mice is effective in

accelerating the onset of puberty in females relative to

those treated similarly but not exposed to the male urine.

Dominant males release an acceleratory substance in their

urine whereas subordinate males do not (Drickamer, 1983a).

Similarly, prepubertal or castrated males do not release an

acceleratory substance in their urine (Vandenbergh, 1969;

Lombardi et al. 1976). Neither the specific type of housing

condition nor the degree of genetic relatedness appears to

mediate the effectiveness of the male pheromone directly.

Drickamer (1983a) found that increasing the density of males

does not alter the pattern of the chemical's release. In

general, the effectiveness of the male chemosignal in

causing pubertal acceleration in females is robust.

Exposing females to amounts of male urine as small as 0.0001

cc per day effectively advances puberty in mice (Drickamer,

1982b, 1984a).

Despite the chemical's apparent acceleratory strength,

such factors as the season or ratio of light-dark exposure

can affect the female and thus indirectly affect whether the

male's chemosignal advances puberty. The substance appears

most effective in producing acceleration when it is

collected from males during light onset and presented to

females at this same time period (Drickamer, 1982a). The

effectiveness of the male chemosignal appears to vary

because of changes in sensitivity of young female mice to

the substance and not due to differences in the substance

excreted by males (Drickamer, 1986). Both male and female

mice have unique patterns of urine deposition that could

influence the likelihood that puberty will be modulated in

juveniles (Drickamer, 1989a).

Certain social conditions can influence the

effectiveness of the acceleratory substance to induce

puberty in females. Vandenbergh (1967) showed that the

acceleratory effect is greater when males are in physical

contact with females; contact-stimulation appears necessary

for the effect to occur (Drickamer, 1974, 1975b; Bronson &

Maruniak, 1975). The active chemical in male mouse urine

appears to be relatively nonvolatile and under natural

conditions it can influence females for some time after the

male has deposited urine (Drickamer, 1986). The substance

in male urine has been found to be effective in accelerating

puberty within three days of exposure (Colby & Vandenbergh,

1974). Acceleration can be accomplished by exposing females

to either male urine for two hours per day or to an intact

adult male for one hour per day (Drickamer, 1983a). Thus,

these findings show that although the age at which female

house mice reach puberty can be reduced by the actual

presence of an adult male, his presence is not necessary for

the acceleration of puberty.

The male chemosignal appears to have its physiological

influence in females via the vomeronasal-accessory olfactory

system (Drickamer & Assmann, 1981). This system may be a

common pathway affecting puberty modulation in various

species. Ablation of the vomeronasal organ eliminates the

ability of both mice and voles to receive pheromonal signals

(Vandenbergh, 1988; Lepri & Wysocki, 1987). Other work

investigating the physiological processes of male-induced

puberty acceleration has been conducted but will not be

reviewed here (see Reiter, 1982; Carter et al. 1986 for


Puberty Acceleration Associated with Pregnant and/or

Lactating Females

Drickamer and Hoover (1979) first documented that the

urine of pregnant or lactating females accelerates sexual

development in female house mice. It is believed that the

active chemosignal is the same that is produced by females

in both reproductive conditions, because a number of

experiments revealed no clear differences between the

chemosignals (Drickamer 1986). Three days of exposure to

urine collected from either pregnant or lactating females is

necessary for puberty acceleration to occur in females that

are less than 30 days old (Drickamer, 1984b). However, the

substance does not cause acceleration in females when it is

presented during the winter months. This lack of an

acceleration in puberty appears due to differences in the

sensitivity of young females to the cues that correspond to

changes in the season (Drickamer, 1986). The acceleration

of puberty does not appear to differ as a function of

kinship among females (Drickamer, 1984c).

Under certain conditions, urine from reproductively

active females does not cause puberty acceleration in

females. For instance, when reproductively active females

are caged in groups or housed with non-reproductively active

females, their urine becomes ineffective in causing either

acceleration or delay in recipient females (Drickamer,

1983b). If young female mice are exposed to urine from both

reproductively active females and urine from group-caged,

non-reproductively active females, the exposed females are

delayed in reaching puberty (Drickamer, 1982c; 1986).

The chemosignals associated with reproductively active

females appear to be relatively volatile and are effective

in causing acceleration when presented in volumes slightly

larger than those of the male signal (0.03 cc of urine per

day; Drickamer & Hoover, 1979; Drickamer 1982b, 1983c).

One difference between the acceleratory chemosignals of

males and those of reproductively active females is that the

chemosignals from females are effective in causing

acceleration regardless of when they are collected or what

time of the day they are presented to females. As noted

previously, the urine from males is most effective in

accelerating puberty when it is collected and presented to

females during the onset of the light portion of the

light-dark cycle (Drickamer, 1982a).

Puberty Acceleration Caused by Females in Estrus

Less is known about how the particular phase of the

estrous cycle influences the chemosignals of the female

donors that can influence puberty in other females.

However, urine from singly-caged female mice in estrus

decreases the latency until puberty is reached in young

female mice. This effect is not seen when urine from

single-caged diestrous females is presented to prepubertal

females (Drickamer 1982c, 1984c).

The chemosignal in urine produced by estrous female

mice is effective in advancing puberty in about 3 days and

is effective when presented in small quantities such as

0.001 cc of urine per day (Drickamer, 1986). No differences

in acceleratory effects were found when urine was collected

from related or unrelated estrous females (Drickamer,


Puberty Inhibition Caused by Group-Housed Females

Only one type of endogenously produced chemosignal has

been found to inhibit or delay puberty reliably in female

mice. Group-housed females produce a substance in their

urine that delays puberty in other females. The time that

estrous smears are detected in young female mice is delayed

when they have been exposed to either soiled bedding of

group-housed females or have had their nares painted with

urine from group-housed females (Drickamer, 1982c).

Females exposed to the urine of group-housed females

will begin to produce the inhibitory substance themselves.

The delay signal's effect occurs later than the effect of

any acceleratory chemosignal. Specifically the delay signal

requires four to seven days of exposure for inhibition to

occur (Drickamer, 1977). Season also influences the degree

of sensitivity young females have to this signal. Females

are not delayed in reaching puberty when exposed to the

substance during the summer, although during other seasons

they are delayed in its presence (Drickamer, 1986).

Puberty Modulation in the Natural Environment

Although the phenomenon of puberty suppression or

advancement is clearly established for house mice in the

laboratory, it is appropriate to question whether similar

processes occur within wild populations. A few studies have

provided evidence that both puberty acceleration and

suppression take place under natural conditions.

Massey and Vandenbergh (1980) conducted a series of

experiments with populations of wild house mice enclosed

within highway cloverleaf sections over a 2-year period.

The urine from the females of two populations was collected

via live-trapping during the spring, when population

densities were low. This urine did not have an effect on

the age of first vaginal estrus of laboratory-housed

females. However, urine collected from the second

population in December, when the population was crowded,

significantly delayed the first estrus in females when

compared with control females. Thus, the researchers

attributed the delay of puberty as being due to the

increased density in the second population.

Urine collected from wild male mice of the highway

"island" populations accelerated puberty in laboratory

females by an average of seven days as measured by age at

first estrus. Puberty acceleration occurred in response to

the male urine despite changes in the season or population

density at the time the urine was collected (Massey &

Vandenbergh, 1981). Thus, it appears that male chemosignals

and subsequent acceleration may be a more prevalent

influence upon puberty in females than the delay chemosignal

produced by female mice under crowded conditions.

In another set of experiments, acute population

explosions were created by Vandenbergh and Coppola (1986)

who introduced 40 second- or third-generation wild female

house mice onto highway islands. This procedure allowed for

a more critical test of the causal relationship between

population density and the release of delay chemosignals.

Urine samples were collected from females on each island

during monthly intervals. Urine samples collected three

weeks after the addition of the females onto the island

populations caused an average delay in first vaginal estrus

of 5.3 days in laboratory-reared females. Thus, this study

provided additional evidence that the puberty-delay

pheromone can be produced by females in response to acute

increases in female density under natural circumstances

(Vandenbergh & Coppola, 1986).

The Social Biology of Voles (Microtus)

A considerable amount of information exists on the

social and mating systems of several species of voles

(Microtus; see Wolff, 1985; Keller, 1985 for reviews). The

social behavior of Microtus is both complex and variable

(Wolff, 1985). Most species are active both during the day

and night, and make use of above-ground runways as well as

burrows. However, there are substantial differences in

patterns of territoriality and of mating system. Below are

listed some of the primary social differences among four

species of Microtus, which are the four species studied in

the following experiments. Broadly, their differences

portray the degree of diversity within the genus Microtus.

Montane voles. Montane voles (M. montanus) are

considered to commonly form a polygynous mating system,

although facultative monogamy may occur at low densities

(Jannett 1980; 1982). Both males and females typically

defend exclusive territories against other same-sexed

individuals (Jannett, 1982). The territories of males

generally overlap the territories of one or more females.

Thus, female montane voles appear typically to mate with

only one familiar male, although no pair bond is formed

(Wolff, 1985). Montane voles become more social and form

aggregations during the winter months (Madison, 1984). The

adult males of the species are typically larger than the

females (males 35% larger; Dewsbury et al. 1980).

Meadow voles. The mating and social system of meadow

voles (M. pennsylvanicus) differ in some respects from

montane voles, and many data have been gathered on their

social behavior (e.g., Madison, 1980, 1984; Webster &

Brooks, 1981). Meadow voles appear to form a promiscuous

mating system in which reproductively active females defend

territories. In contrast to montane voles, the home ranges

of males may overlap those of several other males as well as

those of females (Wolff, 1985). Cases of one female mating

with several males have been reported (e.g., Webster &

Brooks, 1981). Meadow voles form aggregations during the

late fall and winter months as do montane voles (Madison,

1984). The degree of sexual dimorphism in body weight is

also relatively high, with adult males being larger than

females (males 22% larger; Dewsbury, et al. 1980).

Prairie voles. In contrast to montane voles and meadow

voles, prairie voles (M. ochrogaster) appear to be more

social and form a monogamous mating system; a communal

nesting group appears to be the basic year-round social unit

(Getz et al. 1990). Evidence from the laboratory also

suggests monogamy. Both the breeding male and female engage

in extensive parental care of offspring (Getz & Carter,

1980), and male prairie voles failed to demonstrate the

Coolidge effect when presented with a new estrous female

after reaching sexual satiety (Gray & Dewsbury, 1973). Both

males and females appear to be territorial and are

aggressive to animals of the opposite sex. Both sexes of

breeding pairs defend a common home range that is

approximately the same size (Gaulin & FitzGerald, 1988).

The degree of sexual dimorphism in body weight is reduced in

comparison to the dimorphism in montane voles and meadow

voles, although the male is somewhat larger than the female

(males 17% larger; Dewsbury et al. 1980).

Pine voles. Pine voles (M. pinetorum) also appear to

be a highly social species and appear to live in communal

groups (FitzGerald & Madison, 1983; Schadler, 1990). The

mating system of pine voles appears to be monogamous,

although cooperative polyandry has been suggested as an

alternative system (FitzGerald & Madison, 1983). However,

more recent evidence suggests that breeding is commonly

restricted to the founding parents of a communal group

(Schadler, 1990). The existence of a monogamous mating

system had also been predicted to be most likely for pine

voles among eight species of Microtus reviewed by Dewsbury

(1981). Other evidence of monogamy includes male

participation in the rearing of young, including the

retrieval and brooding of infants (Shadler, 1990; Oliveras &

Novak, 1986).

In contrast to many other species of voles, pine voles

are almost entirely fossorial, and come to the surface only

occasionally to feed (Wolff, 1985). Pine voles also appear

to be territorial. FitzGerald and Madison (1983) found that

each family had a discrete non-overlapping territory from

other family groups. Differences in body weight between the

sexes appear minimal in pine voles (males 2% smaller;

Dewsbury, 1990).

Puberty Modulation in Voles (Microtus)

Although several researchers have clearly demonstrated

that puberty delay and acceleration occur in a few species

of Microtus, such as prairie voles (M. ochrogaster; Carter

et al. 1986) and California voles (M. californicus; Batzli

et al. 1977), others have made weaker claims that such

processes do or do not occur in other species of Microtus.

For example, Jannett (1978) referred to field evidence that

suppression occurs in montane voles (M. montanus) and Batzli

et al. (1977) claimed that suppression does not normally

occur in meadow voles (M. pennsylvanicus). Below, I review

the primary literature regarding the four species of

Microtus that were used in the later series of experiments.

Prairie voles. Most research concerning puberty

modulation in Microtus has been conducted with prairie

voles, where the presence of puberty suppression and

acceleration are strongly supported (see Carter et al, 1986

for review). In an early study that indicated puberty delay

occurs in prairie voles, Hasler & Nalbandov (1974) examined

pairs of weanling females caged with males with different

characteristics. Weanling females that were caged with a

littermate male had significantly longer latencies until

vaginal opening and production of a first litter than

compared to females that were caged with either a

non-littermate male or an adult male. The range of days

until vaginal opening occurred among these groups was

substantial. Females kept with littermate males became

perforate 30 days after pairing, on average, compared to

females paired with nonlittermate males that became

perforate eight days after pairing.

Batzli et al. (1977) found evidence that both sexes of

prairie voles could be suppressed developmentally, as

assessed by differences in body weight, when they were

housed with littermates. During this period, females

remained vaginally imperforate and males had abdominal

testes. After siblings were paired with unfamiliar adults

of the opposite sex, rapid increases in body weight and

subsequent reproduction occurred.

The stimuli necessary for the acceleration of puberty

in prairie voles have been studied. Young female prairie

voles, when exposed to a sexually experienced male for a 1 h

period, showed significant increases in uterine weight as

soon as 2 days after exposure (Carter et al. 1980). Drops

of male urine applied to the upper lip of young females

caused significant increases in uterine weight compared with

females caged alone, with a female sibling, a castrated

male, or exposed to the urine of a castrated male.

In another series of experiments, kinship per se was

shown not to be a limiting factor for the advancement of

puberty in prairie voles (Carter et al. 1980). When urine

from male siblings was applied to the nares of females for 6

consecutive days, it led to significantly heavier uterine

weight in females, compared to females housed with either a

sibling male or with a sibling male and treated with water

placed on the nares.

Together, the results of studies with prairie voles

suggest that a chemical agent or pheromone present in male

urine can be passed from male to female by direct contact.

From behavioral observations, Carter et al. (1980) suggested

that the active male pheromone was transmitted by

naso-genital contact between the sexes. However, sibling

prairie voles rarely engage in naso-genital investigation

and thus the lack of investigation between siblings may

function as a barrier to reproductive activation and

incestuous matings (Carter et al. 1986).

Although it is not clear how siblings inhibit

reproduction in other siblings, young female prairie voles

appear to be a primary source of reproductive suppression

for other male and female siblings. Getz et al. (1983)

found that for 15 suppressed litters, all but 1 of 32

females (or 97%) reared with sibling females were

suppressed, while only 3 of 15 (20%) reared with sibling

males were suppressed. A similar finding was apparent in

males; 10 of 11 males (91%) reared with sibling females were

suppressed and only 3 of 9 males (33%) raised with sibling

males were suppressed. The results suggest that for prairie

voles, the sex of siblings can have a substantial effect

upon the timing of puberty.

Additional work with prairie voles has revealed that

the suppressive effect of female presence appears largely

due to chemosignals present in their urine (Getz et al.

1983). In this study, virgin prairie voles were first

reproductively stimulated by exposing them to an unfamiliar

sexually experienced male for a one-hour period, then the

females were housed in a variety of conditions. The uteri

were weighed 48 hours later. The uterine weights of females

that had urine from either a female sibling, a virgin

non-sibling, or a pregnant female, placed on their nares did

not differ significantly from the uterine weights of

non-stimulated females. The only significant increase in

uterine weight was in the group of females that were

stimulated by the male and then maintained alone. Thus, the

results indicate that urine from female prairie voles is

effective in counteracting or suppressing puberty in female

prairie voles.

Although it is not known how the chemosignal is

transmitted between females, activation by the non-volatile

male acceleratory pheromone appears to be caused by mutual

anogenital investigation between the sexes (Carter et al.

1980). Hofmann and Getz (1988) found that virgin prairie

voles that were exposed frequently to unfamiliar males could

"override" the reproductive suppression typically

experienced by females that remained within a family group.

Thus, it seems plausible that under natural conditions where

density is dramatically increased, such as during the growth

phase of a population cycle, normal suppression can be

counteracted by male presence. In support of this argument,

Getz and Hofmann (1986) found that of free-ranging prairie

vole females that remained at the natal nest at low

population density, 18% were reproductively active, while

77% became reproductively active at high density. All

females that dispersed from the natal nest were found to

became reproductively active.

Pine voles. Both puberty delay and acceleration appear

to occur in pine voles, M. pinetorum (Schadler, 1983; Lepri

& Vandenbergh, 1986). Schadler (1983) presented evidence

that male siblings are an important source of reproductive

inhibition for female siblings. Mating was inhibited

between a female and an unrelated male, while a male sibling

was sequestered behind a wire mesh barrier. Other evidence

of puberty delay was shown by Lepri and Vandenbergh (1986)

when they placed young female pine voles with adult males

into two type of cages: one with clean bedding or another

with bedding that had been soiled for two weeks by the

female's family. Forty-eight hours later, the females'

uteri and ovaries were removed and weighed. The uteri and

ovaries of those placed in the clean cage were significantly

heavier than those placed in the cage soiled by the family.

In another experiment, Lepri and Vandenbergh (1986)

demonstrated puberty acceleration in pine voles by exposing

4-week-old females to feces and urine from animals in one of

the following groups: (1) intact males, (2) castrated males,

(3) singly-housed females, (4) group-housed females, or (5)

control (clean cage). When uterine and ovarian weights were

compared, results indicated significant increases in both

organs from females exposed to the stimuli from the males of

either groups compared those from the control females. No

significant differences were found when the organs of the

control group were compared to those in the two groups that

received female stimuli.

Meadow voles. Although there is evidence that pubertal

acceleration occurs in female meadow voles (Baddaloo &

Clulow, 1981), the evidence is equivocal for any type of

puberty delay. Pasley & McKinney (1973) presented evidence

that females caged in groups of eight had smaller ovaries

and uteri than singly-housed females. However, it is

possible that this type of sexual suppression was due to

overcrowding and stress and not due to pheromones. Batzli

et al. (1977) reported they did not find suppression to

occur in five of six litter-housed groups of meadow voles.

Additional data are needed to determine if sexual

suppression occurs in meadow voles as a result of pheromones

and not due to overcrowding and stress.

Montane voles. Fewer reports of pubertal acceleration

or delay can be found scattered in laboratory and field

reports for other species of Microtus. Field evidence

suggests that pubertal delay occurs in montane voles

(Jannett, 1978), although a critical test of this phenomenon

has not been reported. Recent laboratory evidence has shown

that puberty acceleration occurs in female montane voles

when exposed to males. Sawrey and Dewsbury (1991) found

shorter latencies until vaginal perforation and first

cornified estrouss) smears in females housed across a

wire-mesh barrier from males, as compared with females

without a male present. Controlled studies are needed to

determine if puberty delay occurs in montane voles when they

are exposed to pheromones.

It is possible that not all species of Microtus show

both delay and acceleration of puberty, or at least are not

affected to the same degree by comparable stimuli. Evidence

suggests that suppression does not occur, or occurs only

weakly, in female tundra voles (M. oeconomus) when they are

housed with male siblings (Facemire & Batzli, 1983). Batzli

et al. (1977) reported morphological differences between

California voles and prairie voles. California voles that

were housed with littermates grew more slowly than controls,

although females became perforate and males became scrotal

at about the same time in the littermate group as did the

controls (30 days). This pattern differed from prairie

voles in which males typically developed abdominal testes

and females remained vaginally imperforate when littermates

were held together. In both species, however, reproduction

by littermates was usually delayed until they were paired

with unfamiliar animals. Thus, there may be a variety of

physiological and behavioral differences related to puberty

modulation among various species of Microtus. The

illumination of the similarities and differences in patterns

of puberty modulation may prove instrumental in

understanding the dynamics of their social and mating


Puberty Modulation in Male Voles (Microtus)

Considerably less research has been conducted on the

various factors that can influence puberty modulation in

male Microtus, although cues from males may be critical for

influencing puberty in other siblings among some species.

Evidence for California voles suggests that male siblings

influence the sexual development of one another. Batzli et

al. (1977) found that the growth rates of males were

suppressed when they were paired with another male sibling,

although growth rates were not affected when they were

paired with a female, a male non-sibling, or with a female


Other evidence suggests that the odors from family

members of California voles suppress male pubertal

development. In this species, the odors from mature males

or sires did not appear either to accelerate or inhibit the

sexual development of males (Rissman et al. 1984). However,

male California voles that were reared in the presence of

family bedding material and subsequently paired with another

non-relative female failed to cause increases in uterine

weight within four days (Rissman & Johnston, 1985). They

suggested that the lack of stimulation in the females was

due to low levels of circulating androgen in the males.

Thus, odors associated with a reproductively active female

might be one source of pubertal suppression in male

California voles.

A few anecdotal accounts of puberty modulation among

males of other species of Microtus exist. For example,

juvenile male common voles (M. arvalis) have been reported

to show reduced testicular development when housed near

cages with crowded adults (Lecyk, 1967). In summary, it

appears that although males of various species of Microtus

may be reproductively modulated by specific stimuli, the

limited data make conclusions premature. There is a paucity

of information regarding pubertal modulation in male voles,

and for males of other rodent species as well. It seems

worthwhile to correct this bias with future studies.

Principles Underlying Puberty Modulation in House Mice

and Voles

There has been little effort to integrate the large

body of literature into an effective theoretical framework

to study the relationships between primer pheromones, age at

first reproduction, and demographics (Vandenbergh & Coppola,

1986). Many of the existing hypotheses regarding puberty

modulation are based upon studies of house mice (Mus

musculus) because most of the research has been conducted

with this species. Vandenbergh and Coppola (1986) suggested

that some inferences may be applied to other species, such

as voles. However, they caution that given the differences

in the reproductive biology among genera, it seems unlikely

that the selective pressures influencing puberty modulation

will be identical across them. Observed similarities among

genera or species must be interpreted carefully when

attempting to formulate general principles.

Below I list some of the most prominent frameworks that

appear useful for integrating our understanding of the

causes and functions of the pheromonal effects upon puberty

modulation found among species such as house mice and voles.

A Life-History Theory of Puberty Modulating Pheromones

One of the broadest theoretical frameworks to

investigate and interpret patterns of puberty modulation is

the life history theory proposed by Vandenbergh & Coppola

(1986). According to this approach, life history tactics

are evolved sets of coadapted traits designed to solve

particular ecological problems (Stearns, 1976). Whether

animals typically reach sexual maturity relatively early or

late, all are viewed to have been selected to maximize

lifetime reproductive success.

Selective factors favoring puberty acceleration.

Life-history theory predicts that for increasing

populations, or those with large fluctuations or repeated

episodes of colonization, early maturity will be favored

(Vandenbergh & Coppola, 1986). Such characteristics

describe feral house mouse populations. In general, it is

believed that selection will favor early and total

investment by individuals to produce the maximum number of

young whenever the environment is highly favorable for

reproduction. They note that previous theories (e.g., Cole,

1954; Lewontin, 1965) have indicated that in a rapidly

expanding population, the age at first reproduction should

be driven to the physiological minimum by natural selection.

Species that possess a combination of life-history traits

including an early age of first reproduction, the production

of many young, and semelparity have been classified as being

"r-selected" (MacArthur & Wilson, 1967; Pianka, 1970). This

combination of traits is often present in species that

experience rapid population growth in favorable


Several factors are thought to favor early

reproduction (Vandenbergh & Coppola, 1986). First, early

reproduction will be favored as reproductive costs decrease

(Schaffer & Elson, 1975). Reproductive cost is the

deleterious effect of present reproduction on future

survival and/or fecundity. Early reproduction will also be

favored when the reproductive value of individuals decrease

as they grow older (Gadgil & Bossert, 1970). These patterns

suggest that early reproduction will be favored when it does

not negatively influence a female's ability to reproduce

later in life and thus lower lifetime reproductive success.

Delayed reproduction (puberty) in house mice. The

function of delayed reproduction or puberty is more obscure

than the function of puberty acceleration (Vandenbergh &

Coppola, 1986). They suggest that it is difficult to

explain why opportunistic species, such as house mice,

benefit from delaying puberty and reproduction. However,

whatever the functions) of puberty delay, it has been found

in several mammalian species (Vandenbergh & Coppola, 1986).

There appear to be general conditions that favor

delayed reproduction. Delayed reproduction could evolve if

individuals gained fecundity or produced better quality

offspring (Vandenbergh & Coppola, 1986). In general, it is

believed that many of the demographic or environmental

factors that favor delayed reproduction are the opposite of

those favoring early reproduction. Some have suggested that

delayed reproduction would be selected in stable populations

at or near the carrying capacity of the environment (Cole,

1954; Lewontin, 1965) or in declining populations (Hamilton,

1966; Mertz, 1971). Selection in saturated environments,

which favors the ability to compete and avoid predators, has

been referred to as "K-selection" (MacArthur & Wilson, 1967;

Pianka, 1970). Traits correlated with this type of

selection often include late maturity, the production of few

and large offspring, long life spans, and extended parental


There are a few causal factors that are thought to

underlie delayed reproduction (Vandenbergh & Coppola, 1986).

For example, as the reproductive costs increase, in terms of

adult mortality, and as the reproductive value an individual

can accrue by not reproducing increases with age, delayed

reproduction will be favored. If reproductive success is

contingent upon age, size, or social status, delayed

reproduction is also favored (Geist, 1971).

Despite the general conditions that have been proposed

to predict under what circumstances early or late puberty

will occur, the current state of life-history theory allows

us to draw only rather vague and imprecise conclusions about

the timing of reproductive maturity (Vandenbergh & Coppola,

1986). Despite this drawback, life-history theory provides

a broad theoretical background from which to interpret new

information. In addition to the previous conditions which

favor puberty acceleration or delay, there are a few

additional principles that may guide our understanding of

these processes. Below, I summarize two of the most

prominent themes which have emerged.

Puberty Modulation (Mutualism (Cooperation) versus Conflict)

One assumption that underlies most explanations for the

evolution of puberty modulation is that puberty modulation

is either of mutual benefit to the sender and receiver of

pheromones or that the sender and receiver of the pheromones

are in some form of conflict.

Examples of puberty modulation in the context of

cooperation. Bronson (1979) proposed that adult male mice

and adult or prepubertal females both gain in fitness

through mutual stimulation of reproductive acceleration via

pheromonal transfer. In this hypothesis, male urinary

pheromones cause the release of LH in recipient females that

speeds the attainment of puberty and ovulation.

Reciprocally, female pheromones cause increases in LH in

males which ultimately leads to increases in testosterone

and additional pheromone synthesis in males. Such a system

could greatly enhance the speed at which males and females

could reproduce, thus benefiting both sexes of breeding


A second proposed benefit of mutualism was raised by

Vandenbergh and Coppola (1986) who proposed that the

detection of the puberty-delay and acceleratory pheromones

produced by adult female house mice provides information to

young females about the general quality of the environment

for reproduction. Presumably under crowded conditions,

adult females release puberty delay pheromones that benefit

both the sender and receiver to postpone reproduction. In

more favorable environments for reproduction, the release of

pheromones that accelerate puberty by females would hasten

the speed at which puberty and reproduction would occur

among female offspring. Presumably, the young females,

their mates, and kin would benefit from more optimally timed


A third example where mutual benefits may occur between

the pheromone senders and receivers is between parents and

offspring in some cooperatively breeding species (Emlen,

1984). Cooperative breeding refers to any situation when

more than two individuals provide care in the rearing of

young (Emlen, 1984). In this example, puberty delay and

inbreeding avoidance may have been co-evolved traits in

species where cooperative breeding occurs.

It appears that certain ecological constraints, such as

a saturated habitat, favor the retention of subadult

individuals within a family group, at least until conditions

enable independent reproduction. Emlen (1984) suggested it

is possible that when the probability of successful

dispersal and independent breeding are low, average fitness

may be increased by remaining in a group until group size

reaches some optimum. Other benefits that may occur in

cooperatively breeding groups include: (1) benefits to

helpers that gain breeding experience; (2) inheritance of

the parental territory; (3) dispersion in groups when

competition for reproductive vacancies is strong; and (4)

some form of reciprocity may take place, such as when one

individual forgoes breeding and helps another rear offspring

with likelihood of return aid at a later point in time; and

(5) increased inclusive fitness through aiding in the

rearing of close relatives (Emlen, 1984). Thus, both the

direct and indirect components of inclusive fitness might be

increased via reproductive suppression when accompanied by

helping behavior.

A final proposal for mutualistic benefit of

reproductive suppression, or the lack thereof, in voles has

been proposed by Christian (1970). Christian (1970)

emphasized the selective pressures exerted by the habitat

where a particular species evolved. Species such as meadow

voles, which evolved in habitats that were patchy and

ephemeral, such as moist meadows, evolved mechanisms that

aided in the colonization of newly created habitats. Thus,

mechanisms that led to successful dispersal were adaptive

for all members. He proposed that a density-dependent

endocrine response that led to increased aggression with

subsequent dispersal would serve this function. Hence, a

lack of sexual suppression among siblings would be expected

in species that evolved under these circumstances.

In contrast, species such as prairie voles that evolved

in the continuous and extensive habitats of the great plains

of central North America were believed to have evolved

mechanisms that enabled a greater level of social tolerance

and be more sensitive to reproductive inhibition (Christian,

1970). It is plausible that mechanisms to inhibit

incestuous mating among family members might be most evident

in highly social species.

Puberty modulation in situations of conflict.

Different researchers have proposed instances where there

appear to be conflicts of interest between pheromone senders

and receivers. Bronson (1979) proposed there was an

antagonism between female house mice to inhibit puberty and

reproduction. Such antagonism between females may be

accomplished either directly through aggression or through

the production of delay pheromones.

Wasser and Barash (1983) proposed a similar hypothesis.

They stressed the role of female-female competition to

reproduce as a key driving force to explain the high rates

of general reproductive failure among the females of many

species of mammals. The antagonistic pheromonal influence

of reproduction between female prairie voles conforms to

their theory (Getz et al. 1983)

Competition among the males of some species via

pheromones may also be present in some species. Although

Bronson (1979) noted the high rate of aggression observed

between territorial male house mice and other males, he did

not discuss pheromonal interactions among them. Vandenbergh

(1971) found that adult male house mice had an inhibitory

effect on the reproductive development in young males, while

the presence of adult females accelerated their development.

The possibility that males may inhibit puberty and

reproduction of other males in other species through

pheromonal communication should remain open for continued


Socially-Dependent Versus Socially-Independent Systems

Another framework within which to view and investigate

the reproductive influences of pheromones on the timing of

puberty can be called the socially-dependent versus

socially-independent dichotomy. The essence of this

framework is that some species of Microtus appear to be more

dependent upon the direct exposure of other animals in order

to become reproductively mature and active. Taylor

(1990/1991) characterized two types of estrus induction

patterns among females of different species of Microtus. He

provided evidence that the highly social prairie voles,

could be characterized as being male-dependent. Females

must typically be exposed to stimuli from males for a

relatively long period of time before estrus is attained.

In contrast, other less social species, such as meadow voles

and montane voles, were referred to as being

male-independent (Taylor 1990/1991). Species of this type

do not attain estrus totally independent of male

stimulation, but rather are much less dependent on direct

male contact than are male-dependent species.

Puberty Modulation as an Artifact

An alternative viewpoint of the phenomena of puberty

modulation is that the effects are some form of laboratory

artifact. Examples of puberty modulation must be evaluated

for the possibility that differences in the timing of

puberty could be the result of close confinement with other

animals, unnaturally high densities, or a product of

artificial selection (see Bronson, 1979; Vandenbergh and

Coppola, 1986).

Bronson (1979) suggested that while the mechanisms

underlying the phenomena in question may be real, it is

possible that the mechanisms evolved to serve one adaptive

purpose in the wild, yet find expression in other ways in

the laboratory environment. Vandenbergh and Coppola (1986)

have argued against the proposal that puberty modulation is

some form of laboratory artifact in house mice. First, they

suggested that the delay of puberty in female mice which

results from a specific stimulus such as a urinary cue

suggests an evolved signalling function that must have some

adaptive value. A second reason is that the general social

context in which puberty delay occurs in the laboratory is

known to occur in the field. Descriptions of the social

organization of commensal mice generally fit the conditions

necessary for pubertal suppression to occur. The last line

of evidence comes from the field studies conducted with wild

house mice confined to clover-leaf highway populations

(Massey & Vandenbergh 1980; 1981, see prior section). These

studies show that at least under some conditions, the

production of delay and acceleratory pheromones can occur in

animals from free-ranging environments. Nevertheless,

additional studies and further demonstrations that puberty

modulation occurs among mice and other species such as voles

in the field or under semi-natural conditions seem


Summary of Principles of Puberty Modulation in House Mice

and Voles

From the primary findings and related principles

presented for puberty modulation in species such as house

mice and voles, it is clear there is no all-encompassing

theory that leads to clear predictions when puberty should

and should not occur, or to the specific stimuli that should

cause modulation, and the specific functions) it serves.

However, the emerging picture is that a variety of stimuli

are capable of influencing puberty and multiple functions

might be served by puberty modulation among various species.

Vandenbergh and Coppola (1986) suggested that discovering

the role of priming pheromones in the interactive process of

life-history and behavioral adaptations will require the

"melding" of empirical and theoretical points of view.

Problems with Previous Studies of Puberty Modulation

with Voles

Scattered reports indicate that puberty modulation

occurs in a variety of species of Microtus. Some species

have been studied in considerable detail (e.g., prairie

voles, see Carter et al. 1986). However, several sources of

confusion have hampered our ability to explore possible

relationships between puberty modulation and the expressed

differences in the social and mating systems among Microtus.

First, the lack of common procedures and measures used by

various researchers appear to be significant, although

reasonably easy, problems to correct. A variety of measures

and procedures have been used to assess the timing of

puberty, ranging from the measurement of body weight to

analyses of hormone binding sites within the brain. Such

diversity of procedures make meaningful comparisons among

species difficult. For example, Dewsbury (1981) attempted

to evaluate the usefulness of several proposed correlates of

monogamy to predict its presence in several muroid rodents.

However, he deemed it was not possible to compare

meaningfully the existing reports for patterns of sexual

maturation across several species. Thus, with such

variation evident for sexual maturation among muroid

rodents, future studies with identical procedures appear

necessary to make useful comparisons among species.

A second problem is the lack of proper controls that

are necessary to identify the effective stimuli influencing

puberty among species. Cues such as the presence or absence

of an adult male or sire, the presence and number of

opposite-sexed siblings, and the presence or absence of a

reproductively active female have all been found to be

effective stimuli that can influence the timing of puberty

in one or more muroid species. Unfortunately, in many

studies such cues are not systematically controlled or

reported. The use of soiled bedding that is transferred

from one cage to another provides one means of critically

testing the assumption that pheromones produced by one or

more individuals cause puberty modulation in others. Such a

procedure controls for the potential influence that

nonolfactory stimuli, such as visual or auditory, could have

upon puberty modulation.

Finally, the database, which is a prerequisite to

determine the general patterns or principles of puberty

modulation, is still relatively small and, in some cases,

misrepresentative of the genus Microtus. For example,

although only a few studies have provided evidence of

puberty modulation in some species of Microtus such as

montane voles (e.g., Sawrey & Dewsbury, 1991), substantial

data exist for other species, such as prairie voles (Carter

et al 1986; Getz et al 1983). In addition, most studies on

Microtus, like those for mice (Mus), are female-biased.

Most studies have been designed to determine how olfactory

and social cues affect puberty in females rather than males

of various species. Ideally, the factors influencing the

onset of puberty would be studied concurrently for both

sexes. Such patterns should be viewed for the possibility

of significant interactions occurring between the sexes of a

given species.

Thus, whereas the present list of problems encountered

with studies of puberty modulation in Microtus is not

exhaustive, it outlines significant sources of ambiguity and

deficiencies in prior studies. Fortunately, with standard

procedures and proper controls used with a number of

species, a greater understanding of how puberty modulation

can impact the formation of contrasting social and mating

systems among Microtus might be possible.


Four species of voles (Microtus) were studied in each

of three experiments. Species studied included pine voles

(Microtus pinetorum), prairie voles (M. ochrogaster), meadow

voles (M. pennsylvanicus), and montane voles (M. montanus).

Breeding colonies of each species were maintained in the

Psychology Building at the University of Florida. These

facilities were accredited by the American Association for

Accreditation of Laboratory Animal Care (A.A.A.L.A.C.).

All subjects were born to breeding pairs that were

laboratory-reared and that had been derived from wild

populations within the United States. Efforts were made to

maintain genetic diversity while guarding against possible

inbreeding in all species. All species were kept in

separate colonies that were housed in windowless and

air-conditioned rooms that were maintained on a reversed

16:8 light-dark photoperiod with light onset at 2000 hr.

All colonies contained animals of both sexes throughout the

experiments on a combination diet of Rabbit Chow (Purina

Mills) and Laboratory Rodent chow #5001 (Purina Mills) with

water available ad libitum. In an effort to promote

continuous breeding and a sufficient supply of subjects, a

handful of lettuce was given to breeding pairs on a weekly

basis. Breeding animals used in Experiment 3 similarly


received these weekly supplements, although no subjects used

in Experiments 1 and 2 received lettuce supplements during

the duration of their test phase.

All subjects were maintained in large 48 X 27 X 13 cm

polycarbonate cages unless they were housed individually, in

which case they were housed in 29 X 19 X 13 cm polycarbonate

cages. All subjects used in the studies were used in only

one experiment.



Experiment 1 was designed to determine if puberty delay

and acceleration occur solely as a function of the presence

of olfactory cues (i.e., pheromones) that are contained in

the soiled bedding in each of four species of voles

Microtus. In several studies where puberty modulation has

been reported, researchers have either housed animals

directly with or across from other animals (e.g., caging

juvenile females with, or across a wire-mesh screen from,

adult males). Although olfactory cues and pheromones are

believed to be key stimuli that produce changes in the

timing of puberty, many procedures have not been designed to

exclude the possibility that other stimuli critically affect

differences in the timing of puberty (e.g., visual,

auditory, or somatosensory cues).

The use of soiled bedding, which is transferred between

donor and recipient animals, has been shown to be one

effective method to expose animals to pheromones while

controlling for exposure to other non-olfactory stimuli that

could influence sexual maturation. Soiled bedding has been

used successfully to reveal changes in reproductive

development among house mice (Drickamer, 1982c), pine voles

(Lepri & Vandenbergh, 1986), and California voles (Rissmann

et al. 1984; Rissmann & Johnston, 1985).

Measures and Predictions

Specific changes in the physiology of male voles that

would indicate sexual suppression would include reproductive

organs that weighed less than the organs of subjects exposed

to the clean bedding. In addition, the anogenital distance

among males considered suppressed would be smaller than the

anogenital distance in those exposed to clean bedding.

Measures that would reflect sexual suppression in female

voles, would include delayed latency until vaginal

perforation and smaller percentages of cornified cells in

the vaginal smears compared to those exposed to clean

bedding. Among several species of voles, higher proportions

of cornified cells are associated with a higher incidence of

estrus and sexual receptivity (Sawrey & Dewsbury, 1985;

Taylor et al. in press). In contrast to indices of sexual

suppression, indices of sexual advancement or acceleration

would include the opposite changes to those listed above for

sexual suppression.

Because of prior evidence that the highly social female

pine voles and prairie voles typically require direct male

contact for full reproductive activation (e.g., Carter et

al. 1987; Schadler & Butterstein, 1979), I predicted these

species would not be affected, or only minimally, when

exposed to the soiled bedding material. These species may

be considered socially dependent for reproductive activation

(i.e., socially-dependent or "male-dependent" species, see

Taylor, 1990/1991).

In contrast, the females of the less social species

(i.e., socially-independent species), meadow voles and

montane voles, have been shown to become sexually receptive

with little or no previous direct exposure to males or to

show marked changes in reproductive physiology through

exposure to male urine alone (Taylor, 1990/1991; Baddaloo &

Clulow, 1981). Field evidence has suggested that both male

and female montane voles are sexually suppressed when they

remain in natal family groups under high densities (Jannett,

1981). Thus, meadow voles and montane voles were predicted

to show marked responses to the presence of the soiled

bedding material. specifically, they were predicted to be

sexually suppressed when exposed to odors from the natal

family or from adults of the same sex, whereas they would be

sexually accelerated when exposed to odors from unfamiliar

opposite-sexed individuals. Young animals not exposed to

odors from others (e.g., clean bedding) were expected to

show intermediate rates of growth relative to the rates of

those in the other conditions.



A total of 514 animals were used in Experiment 1, 256

were male and 258 were female. All were born to existing

laboratory stock maintained at the University of Florida.

The four species included pine voles (Microtus pinetorum),

prairie voles (M. ochrogaster), meadow voles

(M. pennsylvanicus), and montane voles (M. montanus). All

measures were recorded between September 1990 through

November 1991.


Animals from each of the four species of Microtus were

weighed and individually caged in 23 X 19 X 13 cm

polycarbonate cages at three weeks of age (day 21-22).

Individuals were assigned randomly (via a random number

table) to one of four conditions: (1) "Family" subjects

received transfers of soiled bedding from their family group

(groups containing an adult male, female, and subsequent

offspring) every other day; (2) "Male" subjects received

soiled bedding from a pooled sample that was derived from

the cages of five unfamiliar adult males (see additional

details below); (3) "Female" subjects received soiled

bedding from a pooled sample of five unfamiliar adult

females; and (4) "Control" subjects received transfers of

clean wood-chip bedding that had been placed in a vole-free

colony room and exposed to air as the bedding within the

cages of animals had been exposed.

Subsequent offspring born to the breeding females that

supplied the bedding in the Family condition were not

reduced in number and were removed from the group at three

weeks of age (21-22 days old). Additional litters born into

the family groups were treated in the same manner. No more

than two animals of the same sex and derived from the same

litter were used in the same condition.

All subjects were kept in homospecific colony rooms and

maintained as outlined in the general procedures. Animals

in each of the experimental conditions were housed in small

cages and maintained on separate shelves in each of the

colony rooms in order to minimize the exchange of olfactory

cues among subjects in the various conditions.

Bedding transfers and maintenance. Bedding transfers

occurred on the first day of placement into the experiment

(week 3) and then on every other day throughout the 6-week

test period. Bedding samples were collected and distributed

into recipient cages during the first four hours of light

onset (2000-2400 h). Pooled bedding came from the cages of

five unfamiliar, individually-housed adult animals of the

appropriate sex that did not have their bedding removed for

four days. Bedding samples were mixed and transferred into

the cages of subjects in 200 cc volumes with the use of

plastic cups that were washed with mild detergent after each

use. All donor cages were cleaned weekly. Cleaning

involved the replacement of all soiled bedding with clean

bedding, except for the retention of 800 cc of soiled

bedding within each family cage and 400 cc in each

individually-housed animal to maintain some common olfactory

cues available to the residents. Subjects that received

transferred bedding had 200 cc of bedding removed from their

cages on the day of exchange and had 200 cc of the

appropriate type of bedding added to replace the lost


Pooled bedding was used in the "Male" and "Female"

conditions to provide a more uniform stimulus to animals in

these conditions. It seemed plausible that some of the

bedding-donors would produce soiled bedding that differed in

stimulus quality from others. For example, females may have

excreted different amounts of metabolites in their urine as

a result of fluctuating hormone levels. Previous

researchers have used similar pooling methods in behavioral

preference tests with house mice (e.g., Coppola & O'Connell,

1988; Drickamer 1989b).

Physiological and morphological measures. Body weights

were recorded for subjects when they were weaned (day 21-22)

and at weekly intervals until they were 56 days of age (9

weeks). By this time, individuals from each species would

be considered to have reached adult status under standard

laboratory conditions, with the possible exception of pine

voles. Lepri and Vandenbergh (1986) found that the median

age for male pine voles to sire a litter was 57 days (North

Carolina population), whereas the median age for first

conception among the females was 50 days.

Other measures of puberty included the day of vaginal

opening for females and the anogenital distance of males at

weekly intervals. Vaginal smears were taken daily from each

female beginning on the day they first became perforate.

All other subjects, including males, were handled daily in a

similar fashion as were females, as a control procedure for

the effects of handling. During all handling procedures,

subjects were held with individually-assigned vinyl gloves

in order to prevent the transference of odors among


At the end of the 9-week period, subjects were

euthanized and selected organs were removed and weighed to

the nearest 0.1 mg. Organs weighed included the uterus,

ovaries, and adrenal glands of females and the seminal

vesicles, testes, and adrenal glands of males. All

dissections and weighing of organs were conducted by the

author who used uniform procedures. In all cases, efforts

were made by the author to remain blind to the condition of

the subjects prior to the dissection procedure. This was

accomplished by dissecting animals in small groups if

possible. During this procedure, the identification cards

of subjects were placed upside-down and beneath the

individual trays that held the removed organs from each

subject. The organs were covered with moist paper towels in

a uniform fashion. The author would then scramble the

position of the dissection trays just prior to the weighing

procedure. Thus, in most cases, the author was blind to the

experimental condition of the animals during the final

cleaning and weighing procedure.

Statistical Analysis

Analysis of variance procedures (ANOVA's) were used to

assess the effects of treatment on subjects. All data were

transcribed to computer spreadsheets and analyzed with the

CSS: Statistica software program (StatSoft, Inc). Data were

analyzed independently for each species and sex, because of

occasional instances of heterogeneity of variances between

the species. Analyses consisted of either one- or two-way

ANOVA's with the experimental condition comprising the

primary between-subjects factor. Some measures, such as

body weight, were recorded over the course of the study and

were analyzed with the additional repeated-measure factor of

weeks. Paired organ weights were analyzed with the

repeated-measure factor of body location (left versus

right), because of known physical asymmetries among some

paired organs (e.g., Pinter, 1968) or other possible

functional asymmetries (e.g., Clark & Galef, 1990). The

alpha level was held at .05 in all comparisons, and all

comparisons were based on a two-tailed probability.


Results of each of the measures are reported below for

the effect of the experimental conditions separately for

each species. The statistical values of ANOVA's are

reported directly in the text or in specified tables,

although the exact probability values of post-hoc

comparisons (Neuman-Keuls tests) are not reported in order

to streamline the text. Only post-hoc comparisons that were

significantly different (p's < .05) are discussed in the

text, or are clearly specified as not being significant when


Body Weight

Generally, the experimental condition (bedding type)

did not substantially influence body weight among the

species. However, male pine voles were significantly

affected by the condition (see below).

Because only the factor of week was statistically

significant among the other species, simply reflecting

increases in body weight across age, analyses for each

species are located in Table 3-1 (see Appendix A for means

at each week).

Pine voles: males. Post-hoc analyses revealed that

male pine voles in the Family condition (22.71 + .53,

N = 16) and Male condition (22.96 + 1.05, N = 14) weighed

significantly more than those reared in either the Control

condition (21.24 .53, N = 17) or Female condition

(20.64 + .65, N = 16) at week 9 (interaction of condition

and week, F(18, 348) = 1.98, p = .010)(see Figure 3-1). The

main effect of week was significant, F(6, 348) = 600.87,

p = < .001), indicating larger body weights across weeks,

although the main effect of condition was not significant,

F(3, 58) = 1.68, e = .181 (see Appendix A for means at each


Anogenital Distance

The anogenital distances of the male prairie voles,

meadow voles, and pine voles were not substantially

influenced by the condition. However, there was some

indication that anogenital distances among male montane

voles were influenced by the condition (see below).

Because only the factor of week was statistically

significant in the other species, simply reflecting

increases in anogenital distance with increasing age,

analyses for all other species are located in Table 3-2 (see

Appendix B for means at each week).

Montane voles. Although a significant interaction of

anogenital distance by week of measurement was found,

F(18, 360) = 1.84, p = .019, post-hoc comparisons did not

reveal significant differences among any of the groups (see

Table 3-2 for means at week 9 and ANOVA results; complete

means for each species and week are located in Appendix B).

The mean anogenital distance of montane voles in the Control

condition (M = 13.44 .44 mm, N = 16) approached being

significantly larger than compared to that in the Family

condition (M = 12.25 + .31 mm, N = 16 (p = .09). The mean

anogenital distances in the other conditions were

intermediate in value to those in the Control and Family

conditions. The main effect of week was significant,

F(6, 360) = 173.00, p < .001), although the main effect of

condition was not, F(3, 60) = 0.33, p = .798).

Adrenal Weight

Adrenal weights were minimally affected by the

condition among all species and sexes, with the exclusion of

male pine voles (see below). The mean adrenal weights and

analyses for all species, when uncorrected and corrected for

differences in body weight, are located in Table 3-3.

Pine voles: males. The condition did not significantly

influence the adrenal weights of male pine voles, when

uncorrected for differences in body weight, F(3, 59) = 1.26,

E = .296 (see Table 3-3 for means and ANOVA results).

However, the analysis of adrenal weights, corrected for

differences in body weight, revealed significant differences

among the conditions (see Figure 3-2). Post-hoc comparisons

revealed that the adrenals of males in the Female condition

(M = 24.78 + 1.17, N = 16) were significantly heavier than

in the Control condition (M = 21.60 1.11, N = 17) or

Family condition (M = 20.06 + 1.35; N = 16)(main effect of

condition, F(3, 59) = 3.32, p = .026). The adrenals from

males in the Male condition were similar in weight to those

from the Control and Family conditions but failed to differ

significantly from those in the Female condition (M = 21.02

+ 1.09, N = 14).

The adrenals were heavier on the left side of the body

than on the right, when corrected for body weight (left

adrenal: M = 22.49 + .64; right adrenal M = 21.28 + .62),

F(l, 59) = 7.58, p = .008. The interaction of condition and

body location was not statistically significant, F(3, 59) =

0.14, p = .929.

Among all species and sexes, the left adrenals were

significantly heavier than those on the right side of the

body (See Table 3-3 for complete means and analyses).

Testes Weight

Testes Weights were not significantly affected by the

condition within any of the species, whether they were or

were not corrected for differences in body weight. Complete

means and analyses are located in Table 3-4. The only

significant effects were attributed to small asymmetries in

the left and right testis weights within meadow voles and

montane voles (see means and effect of position in Table


Seminal Vesicle Weight

The experimental conditions did not significantly

influence the seminal vesicle weight within any species,

whether they were or were not corrected for differences in

body weight (see complete means and analyses in Table 3-5.

Ovarian Weight

The experimental conditions did not significantly

influence the ovarian weight of any species, whether they

were or were not corrected for differences in body weight

(see means and analyses in Table 3-6).

Uterine Weight

The experimental condition did not significantly

influence the uterine weights among the pine voles, prairie

voles, or meadow voles (see means and analyses for all

species in Table 3-7). However, significant differences

were evident in the uterine weights of the montane voles as

a result of the condition.

Montane voles. The condition significantly influenced

the uterine weights of montane voles (see Figure 3-3). When

uterine weights were compared, without correcting for

differences in body weight, the uteri of females in the

Control condition (M = 27.46 + 1.92) weighed significantly

more than the uteri found in the Family condition (M = 18.56

+ 1.92) and in the Female condition (M = 16.52 + 2.22) (main

effect of condition, F(3, 60) = 4.55, p = .006). The mean

uterine weight among those in the Male condition was

intermediate to those in the other conditions (M = 24.35

+ 2.97) and was not significantly different from them. This

result is the first example, within a controlled

environment, of sexual suppression in female montane voles

that are exposed to the bedding from a family group or from

adult females.

When the analysis of uterine weight was corrected for

differences in body weight among montane voles, the results

were similar to those of the unadjusted analysis (main

effect of condition, F(3, 60) = 4.61, p = .006 (see Figure

3-3). The adjusted uterine weights were significantly

greater in the Control condition (M = 97.71 + 10.65) than in

the Family condition (M = 61.02 + 4.42) or Female condition

(M = 58.92 + 5.74). However, in addition, the females of

the Male condition (M = 91.21 + 14.45) had significantly

heavier uteri than those in the Family condition.

Status of Vaginal Perforation

The total numbers and percentages of females that

became vaginally perforate during the course of the study

are summarized in Table 3-8 for all species. Clear

differences were evident among the species in their modal

pattern of vaginal perforation. Pine voles displayed the

most atypical pattern of vaginal perforation with respect to

the other species. None of the 66 pine voles were perforate

at the beginning of the study (week 3) and only 2 of the 66

(3.0%) became perforate during the 9-week study. This

slight shift in the frequency of pine voles that became

perforate was not significant between weeks 3 and 9 (McNemar

chi-square: X2 = .50, p < .479). Nearly all of the females

of the other species became perforate at some time during

the study (see Table 3-8).

The percentages of females that were perforate at the

beginning of the test were different among the four species,

X2(3, N = 258) = 70.08, p < .001. Post-hoc comparisons

(chi-square) revealed that all species differed

significantly from pine voles in the proportions of females

that were perforate on week 3. However, significantly fewer

prairie voles were perforate at week 3 than were meadow

voles, (X2(1, N = 167) = 18.36, p < .001, and montane voles,

X2(1, N = 161) = 12.18, p < .001. Meadow voles and montane

voles did not differ significantly on this measure,

X2(3, N = 186) = .91, p < .341.

Delay until Vaginal Perforation

Statistical analyses of the delays of vaginal

perforation were problematic, because properly they would be

limited to those females that were not perforate on the

first day of the study (week 3). However, the majority of

all meadow voles (34 of 62 or 55%) and many montane voles

(26 of 64 or 41%) were perforate on the initial day of the

study and would be excluded from the analysis. In contrast,

only 5 of 66 (8%) prairie voles and none of the 66 pine

voles were perforate on the first day of the study. The

total number of subjects and percentages of subjects for

which vaginal smears were obtained are summarized in Tables

3-9 and 3-10 for each species and by condition.

The species differed appreciably in the time at which

they became vaginally perforate. Figure 3-4 displays the

cumulative percentages of females that became perforate

throughout the study as a function of species. Nearly 80%

of the meadow voles became perforate within the first 4

weeks of age, while prairie voles reached the 80% mark

nearly a week later (Day 33). Over 50% of the montane voles

were perforate by day 23, but the cumulative percentage of

all montane voles did not reach the 80% criterion until day

41. Only two pine voles became perforate, the first at 44

days of age and the second on last day of the study (Day


The mean ages (in days) that vaginal perforation

occurred for subjects in each species and condition, that

were imperforate on the first day of the study, are shown in

Figure 3-5. Sample sizes were not uniform across all

species and conditions because of the different patterns of

typical vaginal opening. Total numbers of subjects ranged

from 14-16 for prairie voles, 5-6 for meadow voles, and 8-10

for montane voles among the conditions.

The condition was not found to significantly influence

perforation latency among the prairie voles, (Kruskal-Wallis

ANOVA, prairie voles: H (3, N = 59) = 4.34, p = .23) or

montane voles: H (3, N = 36) = 5.74, p = .12). Meadow voles

were not analyzed statistically because of the small number

of subjects amenable to this analysis (N = 22).

Vaginal Smears

Three types of cells were classified and counted from

the vaginal smears, following the method of Taylor

(1990/1991). The cell types analyzed included cornified

cells, nucleated cells, and leukocytes. Frequencies of each

type were converted to percentages of the total number of

cells in each smear, because of the variability in the total

number of cells obtained per smear. Similar conversions of

cell frequencies have been used previously in studies with

laboratory rats (e.g., McClintock, 1983, 1984) and with

voles (e.g., Sawrey, 1989/1990; Shapiro & Dewsbury, 1990).

Data from female pine voles were excluded from the analysis

because only two became perforate during the study.

All species produced vaginal smears that were typically

dominated by either cornified cells or leukocytes, with

appreciably fewer nucleated cells. These characteristics of

smears were typical of those found previously for several

species of voles (e.g., Sawrey & Dewsbury, 1985; Taylor,

1990/1991). Nucleated cells were relatively few in number

and relatively constant in proportion throughout the

duration of the study. In contrast, cornified cells and

leukocytes were typically in reverse proportion to one

another when changes in the percentages of cells were


Prior to conducting statistical analyses, all cell

percentages were pooled for each subject by two-day block

intervals. This procedure generally reduced daily

variability in the cell percentages across successive days.

Prior to pooling, graphs were made of the data as a function

of each day; no indications of cyclical fluctuations were


Effect of condition: within-species comparisons of cell

types. Two types of analysis were conducted for

within-species comparisons of cell types. The first

analysis consisted of comparing the proportions of each cell

type as a function of the experimental conditions for each

two-day block (Kruskal-Wallis ANOVA by Ranks tests). Thus,

a total of 21 two-day blocks (representing Days 21-22

through 61-62) were analyzed sequentially to determine if

the condition influenced the percentages of cell types. In

the case of a significant result, post-hoc comparisons were

made to identify which conditions differed significantly

(Mann-Whitney U Tests).

In the second analysis, repeated-measures analyses were

used to assess whether significant changes in the

percentages of each cell type occurred as a function of age.

These analyses were done separately for each condition and

species. Cell percentages were analyzed on alternate blocks

of days representing the ages of 33-34 days through 61-62

days (Friedman ANOVA by ranks tests). This reduced sample

of days was necessary due to fewer smears available during

the youngest ages and thus the necessary exclusion of data

from repeated-measures analysis (see Table 3-9). Thus, a

series of eight means was compared in each repeated-measures

analysis. Post-hoc comparisons (Wilcoxon Matched Pairs

Tests) were used to identify where significant changes

occurred across the blocks of days when the Friedman ANOVA

was found to be statistically significant (p's < .05).

Prairie voles: cornified cells: effect of condition.

The percentages of cornified cells from prairie voles are

shown as a function of experimental condition in Figure 3-6

(each mean plotted represents the pooled data of individuals

by two-day block intervals). It was evident that relatively

little variation occurred in the cell percentages as a

function of the condition among prairie voles. A

statistically significant change was identified for only one

of the two-day blocks (Day 27-28: H(3, N = 35) = 8.90,

p = .031). During this block, subjects in the Female

condition had significantly more cornified cells than those

in the Male condition (M's = 27.5% versus 17.9%; total

numbers of subjects can be determined from Table 3-9).

Prairie voles: effect of age. Relatively small changes

in the percentages of cornified cells occurred as a function

of age. Repeated-measures analyses failed to detect any

significant change in the cell percentages for subjects in

any of the four conditions.

Additional comparisons of the percentages of nucleated

cells and leukocytes have been placed in Appendix C. In

general, the proportions of nucleated cells varied little as

a function of the condition or age for all species. Thus,

the proportions of leukocytes often varied inversely to the

proportions of cornified cells and are somewhat redundant

for purposes of analysis.

Meadow voles: cornified cells: effect of condition.

The mean percentages of cornified cells as a function of the

experimental condition are shown in Figure 3-7. Despite

somewhat greater differences among the cell means than those

found in prairie voles, and relatively large sample sizes

(range of total N's = 47-56), no significant differences

were found within any of the blocks of days as a function of

condition. The lack of significant differences also held

true for all comparisons of nucleated cells and leukocytes

(Appendix C).

Meadow voles: effect of age. Repeated measures

analyses revealed significant changes in the percentages of

cornified cells within all four conditions of meadow voles.

There were general increases in the percentages of cornified

cells for subjects in all conditions, although the relative

percentages of cornified cells for the Control condition

were typically the smallest among those from all conditions

(see Figure 3-7; Control condition: X2(7, N = 11) = 31.39,

p < .001). Significant differences occurred among the

following blocks of days for subjects in the Control

condition (Note: differences indicated below are in the

direction of later days being significantly higher in cell

percentages than earlier days except where noted "*"): Days

33-34 versus Days 49-50 through Days 61-62; Days 37-38

versus Days 41-52, 49-50 through Days 61-62; Days 45-46

versus Days 53-54 through Days 61-62; Days 49-50 versus Days

57-58 through Days 61-62.

Significant changes in the percentages of cornified

cells for subjects in the Family condition were evident,

X2(7, N = 11) = 29.85, p < .001), and were as follows: Days

33-34 versus Days 45-46 through Days 57-58; Days 37-38

versus Days 45-46 through Days 61-62; Days 41-42 versus Days

45-46 through Days 61-62.

Significant changes in the percentages of cornified

cells for subjects in the Male condition were present,

X2(7, N = 12) = 36.97, p < .001), were as follows: Days

33-34 versus Days 41-42, Days 49-50 through Days 61-62; Days

37-38 versus Days 41-42, Days 49-50 through Days 61-62; Days

41-42 versus Days 53-54 through Days 61-62; Days 45-46

versus Days 49-50 through Days 61-62.

Significant changes in the percentages of cornified

cells for subjects in the Female condition were present,

X2(7, N = 13) = 52.08, p < .001), were as follows: Days

33-34 versus Days 41-42 through Days 61-62; Days 37-38

versus Days 45-46 through Days 61-62; Days 41-42 versus Days

45-46 through Days 61-62; Days 45-46 versus Days 53-54

through Days 61-62; Days 49-50 versus 57-58.

Montane voles: cornified cells. The mean percentages

of cornified cells among montane voles in the various

conditions varied more as a function of condition than the

percentages observed in prairie voles and meadow voles

(compare Figures 3-8 for montane voles to Figures 3-6, and

3-7). Typically, there were gradual increases in the

percentages of cornified cells throughout the study for

montane voles.

Montane voles: effect of condition. Statistical

differences were found among subjects in the various

conditions on blocks of days including Days 53-54, 57-58,

and 61-62. Among the first and last of these three blocks,

the subjects in the Male and Control conditions had

significantly greater mean percentages of cornified cells

than those in the Female and Family conditions. The

intermediate block (Days 57-58) also differed in the same

manner, except the difference between the Male and Family

condition failed to reach a significant level (see Figure

3-8 for clarification).

Montane voles: effect of age. Repeated measures

analysis revealed significant shifts in the percentages of

cornified cells within each of the four conditions. Montane

voles in the Control condition had significant shifts in the

proportions of cornified cells between the following blocks

of days: Days 33-34 versus 41-42, 49-50 through 61-62; Days

37-38 versus 57-58 through 61-62; Days 41-42 versus 53-54

through 61-62; Days 45-46 versus 49-50 through 61-62; Days

49-50 versus 57-58 through 61-62.

Significant shifts in the percentages of cornified

cells were found for those in the Family condition among the

following blocks of days: Days 33-34 versus 49-50, 57-58

through 61-62; Days 37-38 versus 57-58 through 61-62; Days

41-42 versus 49-50 through 61-62; Days 45-46 versus 61-62;

Days 49-50 versus 61-62; Days 53-54 versus 57-58.

Changes among blocks of days for subjects of the Male

condition were statistically significant among the following

blocks of days: Days 33-34 versus 53-54 through 61-62; Days

37-38 versus 53-54 through 61-62; Days 41-42 versus 49-50

through 61-62; Days 45-46 versus 53-54 through 61-62; and

Days 49-50 versus 53-54, and 61-62.

Finally, significant shifts in the percentages of

cornified cells were identified for subjects in the Female

condition among the following blocks of days: Days 33-34

versus 41-42 through 49-50, and 57-58 through 61-62; Days


37-38 versus 49-50 through 61-62; Days 45-46 versus 61-62;

Days 49-50 versus 61-62; Days 53-54 versus 61-62; and Days

57-58 versus 61-62.


Prairie voles and pine voles were expected to show

fundamentally different patterns of response from those of

the meadow voles and montane voles. I predicted that the

less social meadow voles and montane voles would show

pronounced sexual advancement when exposed to the bedding of

unfamiliar opposite-sexed individuals, and show signs of

sexual suppression when exposed to same-sex odors. Animals

exposed to clean bedding (Control condition) were expected

to have intermediate indices of sexual development compared

to those in the other conditions. In contrast, I believed

there would be little or no differences in sexual

development within the prairie voles or pine voles.

The experimental results of the exposure to the soiled

bedding on general and sexual development are discussed

below for each of the measures.

Body Weight

Male pine voles were the only group that differed

significantly in body weight as a function of the condition.

Males in the Family and Male conditions weighed more than

those in the Control and Female conditions (Figure 3-1).

These results, if repeatable, are contrary to predictions.

The results suggest that the olfactory cues associated with

adult males or family groups cause general increases in the

rate of growth of males exposed to them, when compared to

patterns of growth of males exposed to clean bedding or to

odors from adult females. Unfortunately, it is not possible

to compare these results with those of other studies, as

pine voles have not been investigated in any similar manner.

However, general characteristics of natural populations

of pine voles suggest that male pine voles are not sexually

suppressed when living within family groups and often are in

the presence of at least one other scrotal male (FitzGerald

& Madison, 1983). Thus, limited information on the

free-ranging behavior of pine voles is at least compatible

with the notion that the exposure of males to the odors from

family groups and/or other males in some manner contributes

to increased body weight. One possible mechanism by which

males might experience increased body weight is simply

through increased food intake. Such a mechanism has been

identified that led to increased weight gain in male musk

shrews (Suncus murinus) when exposed to females (Wayne &

Rissman, 1990).

It is not known why pine voles were the only species to

experience differences in body weight as a function of the

condition. Other studies have shown that the body weights

of other species of Microtus can be affected by exposure to

pheromonal cues. Female meadow voles have been shown to

have significantly greater increases in body weight,

compared to controls, when the urine of mature males

trickled directly into the cages of females via small tubes

(Baddaloo & Clulow, 1981). Other research has provided

evidence that male and female prairie voles could differ in

body weight, by nearly 10 g respectively, as a function of

whether they had separate air supplies or shared a common

air supply within the colony (Batzli et al. 1977).

Together, the results of previous studies suggest that

differences in body weight might have been detected within

some of the other species, had the procedures been

different. The use of a more continuous supply of urine and

pheromones or a separate supply of air for each subject may

have led to detectable differences as a function of the

condition. However, given the generally small differences

in body weights among the male pine voles and number of

comparisons made, one must also consider the results were

due to chance alone and not due to pheromonal differences.

Additional study seems warranted to clarify whether these

patterns of weight gain among male pine voles can be

replicated, what mechanism accounts for differences in body

weight, and what functional differences they may be


Anogenital Distance

The anogenital distances among montane voles might have

differed as a function of the conditions, but the results

are ambiguous. Whereas the overall F statistic was

statistically significant, post-hoc comparisons failed to

detect significant differences among the groups. Males in

the Control condition had a larger mean anogenital distance

than those in the Family condition, although it was not

significantly different (E = .09; Neuman-Keuls test). This

difference is in the hypothesized direction, but the

absolute difference between the groups limits making any

firm conclusion.

In an attempt to determine statistically whether the

differences in anogenital distances among montane voles were

systematic effects caused by the conditions, an analysis of

covariance was conducted, using body weight at week 3 as a

covariate. Results were similar to those of the primary

analysis, i.e., the interaction of anogenital distance and

week of measurement was statistically significant, F(18,

360) = 1.84, p = 0.019, although no post-hoc comparisons

were significantly different. Thus, the results of the

anogenital distance comparisons are only suggestive of

differences produced by exposure to the different bedding.

Unfortunately, no similar studies have been conducted with

montane voles, or other species of Microtus, in order to

make any comparisons between studies.

It is not known what functional difference, if any, a

larger anogenital distance would reflect. Presumably, the

measure reflects, in part, the relative size of the testes

which lie proximally between the penis and the anus. In the

present study, the correlation between testes weight and

anogenital distance was relatively small among all montane

voles (N = 64; rg = .31), although it was slightly higher in

the Control males alone (N = 16; rs = .37). Thus,

differences in testis weight do not lend much support for

the possibility that the differences in anogenital distance

largely reflect differences in testis weight. The analysis

of the testes weights of the montane voles did not reveal

any significant influence of the conditions (see below).

Additional study would seem necessary to establish whether

the differences in anogenital distance in montane voles are

repeatable outcomes of exposure to these substrates.

Adrenal Weight

The adrenals differed systematically in weight as a

function of the treatment among male pine voles. The

adrenals, when corrected for differences in body weight,

from males in the Female condition were significantly

heavier than those in the Control and Family conditions.

Because males of the Female condition were significantly

lighter in body weight than those in the Family and Male

conditions, it is possible there is a causal relationship

between increased adrenal activity and decreased body

weight. Prior studies with male meadow voles have shown

that subcutaneous injections of ACTH produced significant

reductions in body weight and concurrent increases in

adrenal weight than among control males (Pasley & Christian,


It is not known what differences there are in bedding

soiled from male and female pine voles. Recent chemical

analysis of volatile compounds in male and female pine vole

urine did not reveal any qualitative difference in the

urinary profiles between them (Boyer et al. 1989). However,

female urine contained three volatiles in higher

concentrations than in male urine, whereas male urine

contained one compound present in higher concentration than

in female urine. Boyer et al. (1989) suggested there may be

differences in the nonvolatile urinary fraction that might

be investigated. Among house mice, the acceleratory

pheromone produced by male house mice appears to be mediated

by a nonvolatile urinary fraction contained in the urine

(Vandenbergh et al. 1975; 1976).

Among all species, the adrenals from the left side of

the body were significantly heavier than those located on

the right. This difference in asymmetry may be common to

all species of Microtus. Pinter (1968) reported that the

adrenals in montane voles were larger on the left than on

the right side of the body, regardless of the sex, age, or

regime of diet or photoperiod. A similar asymmetry in

adrenal weight had been reported for California voles

(M. californicus)(Mullen, 1960). The specific functional

difference of such asymmetry, if any, is not known.

Testes Weight

Comparisons of the testes weights did not indicate any

effect of the experimental condition among any of the

species. It is possible that changes in testicular weight

among male Microtus are relatively unaffected by exposure to

different pheromones, although exposure to stressful

environments may produce substantial changes. Lecyk (1967)

provided evidence that male common voles (M. arvalis), that

were housed adjacent to cages with crowded and sexually

active voles, had lighter testes than control males. Other

indirect evidence that testicular weight can change as a

function of stress was provided by Pasley and Christian

(1971). Male meadow voles, injected with ACTH across a

series of days, had significant decreases in testes weights

when compared to noninjected controls.

It is possible that testis weight is a relatively

insensitive measure of male reproductive activity, at least

when investigating the influence of pheromones or other

olfactory stimuli on reproductive maturation. More precise

measures of reproductive activity might reveal systematic

differences among voles exposed to different pheromones.

Measures such as the level of circulating androgen or

characterization and counts of spermatozoa may be

appropriate. Research with California voles has shown that

titers of androgens were found to be significantly lower in

males exposed to bedding from their mothers than in those

exposed to bedding from unrelated males; testes weights were

not found to differ among these groups (Rissman et al.


Seminal Vesicle Weight

The experimental condition did not have a noticeable

effect on the seminal vesicle weight among any of the

species. Why no significant differences were found is

puzzling. Prior studies with California voles have shown

that differences in seminal vesicle weight can be found when

males are exposed to different types of soiled bedding. The

seminal vesicles of California voles that were exposed to

bedding soiled by their own mother, their family group

(including the sire), or an unrelated mother were

significantly smaller than those of males reared in clean

bedding (Rissman et al. 1984; Rissman & Johnston, 1985). In

contrast, the presentation of soiled bedding from unfamiliar

adult males, or bedding from the family that had been

supplemented with bedding from the separated father,

produced seminal vesicles of similar weight in both groups.

Thus, the data from California voles suggest that the

development of the seminal vesicles can be delayed in young

males when they are exposed to odors from their mothers or

from unrelated mothers, but not delayed by exposure to odors

from unrelated adult males or from their fathers (Rissman et

al. 1984).

Ovarian Weight

The experimental condition did not have a noticeable

effect on the ovarian weight in any species, whether they

were corrected or uncorrected for differences in body

weight. However, previous studies suggest that the ovarian

weight among some species of Microtus can be affected by

exposure to pheromonal stimuli. Baddaloo and Clulow (1981)

found that female meadow voles that were exposed to male

urine, that had been designed to drip into the adjoining

(empty) half of divided cages, had significantly heavier

ovaries than compared to those of control females.

Female pine voles have also been shown to have

increases in ovarian development when exposed to pheromonal

stimuli. Lepri and Vandenbergh (1986) placed individual

females directly below the cages of animals of different

stimulus qualities. This procedure enabled feces and urine

from the animals) in the top cages to fall into the cages

housing the females. Results indicated that the ovaries of

the females exposed to the excreta from either individually

housed intact males or castrated males were significantly

heavier those of females exposed to an empty cage or to the

excreta from singly-housed females or group-housed females.

Uterine Weight

By at least one standard, the uterus is considered to

be the best and most reliable bioassay for circulating

estrogens in female house mice (Bronson & Stetson, 1973).

Female montane voles in the Control condition were found to

have significantly heavier uteri than those in the Family

and Female conditions (Figure 3-3). In addition, the uteri

from those in the Control and Male conditions were also

significantly heavier than those in the Family and Female

conditions when the uteri were corrected for differences in

body weight. These results are the first to demonstrate

sexual suppression of female montane voles when they are

exposed to adult female bedding or family bedding under

controlled conditions. Previous field evidence had only

suggested such a phenomenon (Jannett, 1978). The results

are in general agreement with predictions and complement the

recent finding of sexual acceleration in female montane

voles when they were exposed to male stimuli (Sawrey &

Dewsbury, 1991). However, if the responses of uterine

weight to the pheromones conform to the distinction of

socially-dependent versus independent dichotomy, it is not

clear why meadow voles also did not show differences in

uterine weight when they were exposed to the different

bedding types.

It is possible that many species of Microtus, including

meadow voles, may require both direct exposure to males and

their pheromones for complete reproductive activation. This

type of synergy has been demonstrated in house mice

(Drickamer, 1974; Bronson & Maruniak, 1975), montane voles

(Sawrey & Dewsbury, 1991), prairie voles (Carter et al.

1987), and pine voles (Lepri & Vandenbergh, 1986), although

the specific causes for it are not yet understood. For

example, among female prairie voles, direct contact with

male urine or housing them in male-soiled cages results in

increased uterine weights, but typically does not elicit

behavioral estrus (Carter et al. 1987). Thus, it is

possible that female meadow voles may require more direct

male stimulation in comparison to montane voles. Evidence

found by Taylor (1990/1991) supports this hypothesis, as

nulliparous female meadow voles showed extremely low

copulation rates after being kept behind a wire-mesh barrier

for up to seven days with only 1 h of daily direct

interactions with males.

Characteristics of Vaginal Perforation

Although none of the species differed significantly in

the latency to become vaginally perforate as a function of

exposure to soiled bedding, clear species differences were

evident in the latencies of the species to become perforate

(Figure 3-4). Meadow voles and montane voles became

perforate either before or shortly after weaning (Day 21).

In contrast, prairie voles were somewhat delayed in becoming

perforate. Pine voles were even more extreme, with only two

of the animals becoming perforate during the course of the


The few pine voles that become vaginally perforate

during the course of the study do not seem uncharacteristic

from personal observations and from other reports (e.g.,

Schadler & Butterstein, 1979). It appears that most female

pine voles, whether housed in isolation or with siblings, do

not become perforate until they are placed with an

unfamiliar male or reach a considerably advanced age. In

pilot work for the study presented, 10 adult female pine

voles were examined for vaginal perforation. Whether pine

voles were housed singly, or with other siblings, none was

perforate despite ranging in age between 90 to 120 days.

Earlier work also supports the finding of substantial

delays in reproductive activity among pine voles. Schadler

and Butterstein (1979) found that among female pine voles

that had been paired for breeding with fertility tested

males, the mean age of first conception for the females was

105 days. Thus, it appears that direct and relatively long

exposure to males may be a normal prerequisite for vaginal

perforation and reproductive activation in pine voles, even

if they have reached a considerably advanced age.

Substantial delays among female prairie voles to become

vaginally perforate have also been documented. Richmond &

Conaway (1969b) found that 98% of more than 200 individually

housed females, or those in compatible female groups,

remained in a state of persistent anestrus with imperforate

vaginas between 3 to 5 weeks of age.

Within-species analysis of the perforation latencies

among prairie voles did not suggest they are strongly

affected by the olfactory cues. The overall comparison test

was not statistically significant, despite there being a

relatively large number of imperforate female prairie voles

at the beginning of the study (N's = 14-16 per condition).

Female prairie voles in the Male condition became perforate,

on average, earlier than those in the Control condition (M's

= 33.0 versus 27.5 days)(Figure 3-5).

One of the difficulties with analyzing the conditions'

effect upon perforation latencies among meadow voles and

montane voles was that many females were vaginally perforate

at the beginning of the test and thus excluded from the

formal analysis. Employing an earlier day of weaning did

not seem plausible for all species, although it was

attempted in pilot work. Weaning pine voles at an earlier

age than 21 days often resulted in their deaths within a day

or two following separation. In retrospect, it would seem

possible to wean each species at different ages that

corresponded more closely to their species-typical age for

weaning. Indices such as the cessation of nursing or the

timing of the first day to ingest solid food is typically

earlier for meadow voles and montane voles than they are for

pine voles and prairie voles (McGuire & Novak, 1984; 1986).

By weaning each species at an age that appears most

appropriate for each species, it would be possible to test

more readily how stimuli affected certain reproductive

responses, at least within each species.

Vaginal Cytology

It was expected that the less social species would show

higher proportions of cornified cells when the females were

exposed to male bedding than would females exposed to

bedding in the other conditions. Those in the Family and

Female conditions were predicted to have fewer cornified

cells than those in the Control condition. The patterns of

cornified cells for all species that became vaginally

perforate are shown in Figure 3-6 through Figure 3-8.

The percentages of cornified cells for prairie voles

among the various conditions were similar, and statistically

only one of the two-day blocks revealed a significant

difference among conditions. It seems plausible that this

one significant difference was spurious, because neither the

few preceding nor the few following blocks of days was

statistically significant. In addition, the total number of

multiple-comparisons tests was substantially high. This in

turn would inflate the likelihood of detecting at least one

statistically significant difference among the days

compared. As predicted, female prairie voles revealed

little response to the different bedding types.

Although variation in the cell percentages for meadow

voles suggested an influence of condition, none were

detected statistically (Figure 3-7). Inspection of the

percentages of cornified cells suggests a trend of more

cornified cells in females exposed to adult female odors

than those receiving no odors (Control). A priori, the

direction of this relationship was not expected but suggests

further study because it is possible that meadow voles

experience sexual acceleration when exposed to odors or

pheromones of other adult females. Among house mice, urine

from pregnant and lactating females accelerates sexual

development in female mice (Drickamer & Hoover, 1979), as

does the urine from female mice that are in estrus

(Drickamer 1982c). It seems plausible that among meadow

voles, pheromones contained in the urine of other meadow

voles that are of either sex may cause sexual acceleration

when compared to those that are not exposed to any

pheromonal source, at least under certain conditions.

Results from the analysis of vaginal smears of meadow voles

by Baddaloo and Clulow (1981) are in general agreement with

this prediction.

There is reasonable evidence that the conditions

affected the proportions of cornified cells of the montane

voles, at least during the last 10 days of the study. The

statistical results revealed systematic differences in the

proportions of cells as a function of condition during the

later portion of the study (Figure 3-8). Specifically,

during three of the last five two-day blocks of smears, the

females in the Male and Control conditions had substantially

more cornified cells than those in the Family and Female

conditions. Although there is the possibility that these

relatively few significant results were due to chance,

because of the relatively large number of comparison tests

(21 for each species and condition representing Days 21

through 62), the distribution of the significant effects

suggests a non-random pattern.

The differences among the uterine weights of the

montane voles add additional support for the hypothesis that

the percentages of cornified cells of montane voles reflect

systematic differences as a function of the condition. The

corrected uterine weights were significantly heavier from

females in the Male and Control conditions compared to those

from the Family and Female conditions. Thus, the

percentages of cornified cells appear to reflect these

differences; larger uteri were associated with higher

percentages of cornified cells among the montane voles.

Given the present results, the montane voles appeared

to be the species that was most affected by the exposure to

the pheromones. The results of the current study and of

earlier studies suggest that, even with similar levels of

male exposure, prairie voles and pine voles take appreciably

longer to become sexually receptive than montane voles and

meadow voles, and typically require direct exposure to males

for complete reproductive activation to occur (Carter et al.

1987; Lepri & Vandenbergh, 1986). Thus, results of the

vaginal smear data provide limited support for the

socially-dependent and socially-independent dichotomy. The

less social species, meadow voles and montane voles, appear

to be more sensitive to conspecific pheromonal cues than are

the more social species. Additional study of these species,

using common procedures, may be useful to determine if these

results are valid, and to shed light on possible functional

differences between the species.

Conclusions (Experiment 1)

A major theme that broadly characterizes the results of

Experiment 1 was the absence of an effect of exposure to the

different bedding types among the species. These results

were surprising, considering that a number of studies have

shown that the reproductive physiology of several species of

voles can be influenced by exposure to the same types of

olfactory stimuli or pheromones that were used in the

present experiment (see Puberty Modulation in Voles).

A few studies have shown specifically that the transfer

of chemosignals contained within soiled bedding can

effectively produce changes in reproductive activity in

house mice (Drickamer, 1982c) and in some species of voles.

Lepri and Vandenbergh (1986) showed that exposure to

chemosignals contained within the soiled bedding of family

groups caused reproductive suppression among female pine

voles. Female pine voles were paired with unfamiliar males

and then housed in cages with either clean or family-soiled

bedding for 48 h. Females that were housed on clean bedding

had significantly heavier uteri and ovaries than females

housed on family-soiled bedding.

Soiled bedding has also been shown to affect

reproductive development among male California voles

(Rissman et al. 1984). Males that were reared from weaning

in bedding from their families had significantly lighter

seminal vesicles when they were 45, 55, and 75 days of age

than those reared in clean bedding (Rissman et al. 1984).

Androgen levels were significantly higher among the males

reared in clean bedding versus those reared on the family

bedding on day 45. Together, these studies show that the

method of exposing animals to soiled bedding can be an

effective means to reveal specific changes in reproductive

physiology, at least among some species of muroid rodents.

Several reasons can be proposed as possible

explanations for the lack of substantial differences in most

reproductive measures as a result of the different

conditions. It is possible that pheromones may be minimally

involved, or their activity overestimated, with regulating

the onset of reproduction in all or most species of

Microtus. It is possible that pheromones are substantially

more effective in regulating reproduction in other species,

such as in house mice (e.g., Bronson, 1979; Vandenbergh &

Coppola, 1986). Clearly more comparative research is needed

to test this possibility. Seminatural studies may be an

appropriate method in which to run biologically relevant

experiments, while still enabling the close monitoring of

reproductive development in individuals exposed to

pheromonal sources.

If pheromones are critically involved with influencing

the timing of reproduction among Microtus, there are at

least three methodological reasons that could account for

the relatively few differences found. First, it is possible

that the pervasive odors within the colony rooms effectively

eliminated many of the differences that might have been

found had animals been reared in environments that

controlled for all or most of the extraneous olfactory

stimuli. All animals were reared and tested in colony rooms

where the odors of other conspecifics animals were housed

that varied in sex, age, and reproductive status, although

the subjects had been on separate shelving for each

condition. Some support for this hypothesis is found in the

work of Batzli et al. (1977), who controlled the air supply

to individual animals. Both male and female prairie voles

had substantially elevated gains in body weight across a

three month period as compared to animals that were treated

similarly but shared a common air supply with other prairie

voles. In addition, Sawrey (1989/1990) found differences in

the patterns of vaginal smears of female montane voles that

had been housed either with six other females in a separate

room or had been housed in a larger colony with animals of

mixed sex and age. Vaginal smears revealed that the six

separately housed females had smaller percentages of

cornified cells than smears from females in the large colony

room (11.9% and 29.8% respectively).

A second possible reason for the relatively few

differences found among the conditions is the possibility

that the daily handling procedure was stressful enough to

effectively obscure or eliminate systematic differences.

There is indirect evidence that suggests such a possibility.

Olsen & Seabloom (1973) found that the event of captivity

caused elevated and prolonged secretion of corticosterone in

wild-caught meadow voles. It is known that increased

secretion of adrenal corticoids, androgens, progesterone,

and other steroids are associated with an inhibition of

reproduction (e.g., Christian, 1975). Thus, the daily

handling procedure may have been a significant source of

stress that might have reduced differences in morphological

and physiological growth between animals in the different


A third possible reason for the relatively few

differences among the conditions stems from the experimental

design. It is possible that larger differences would have

been found among the conditions had the soiled bedding and

pheromones been presented to the subjects more frequently

than every other day. Some researches have used procedures

whereby urine was channeled directly into the cages of

subjects via tubes or they have placed cages with wire

bottoms and urine donors directly over the subject's cage

(e.g., Baddaloo & Clulow, 1981; Lepri & Vandenbergh, 1986);

thus, a near-continuous supply of pheromonal odors had been

available. Some of the information gathered in house mice

suggests that some pheromones are relatively volatile and

therefore short-lived in functional activity, while others

appear to be more stable and active over a few days. The

actual chemical composition and properties of the pheromones

from voles remain largely unknown.

Future research that is designed to identify the

effects of exposure to pheromones may be based on the

general design of this study. However, three improvements

in the design could be based on the three possible sources

of largely negative results listed above. Ideally, subjects

would have individual supplies of air, be minimally stressed

through extraneously handling or exposure to other animals,

and have a more continuous exposure to pheromonal sources.

The apparatus of Baddaloo & Clulow (1981) represents a

modification in procedure that might detect physiological


responses from subjects. Despite the few significant

results found in the present study, the results of many

other studies suggest that the comparative study of the

functions of pheromones among species of Microtus, appears

to be a productive area for further advances in the

elaboration of their evolved reproductive and social



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