Seasonal changes in testosterone levels in wild Mexican cottontails Sylvilagus cunicularius

Abstract

We studied serum testosterone levels in the endemic Mexican cottontail, Sylvilagus cunicularius, which has been reported to show seasonal breeding. Animals were trapped in the wild and in a field enclosure in the National Park La Malinche in central Mexico over a period of five years. Serum testosterone (T) levels were quantified by ELISA from blood samples. T levels of adult males were lowest around 4 months after the onset of the annual reproductive season and were already high prior to the onset of breeding. As expected, the T levels of adult females were consistently lower than in males, and there were no differences in T level with respect to female reproductive state. There were no detectable sex-specific differences in juveniles and subadults, but there was a marked increase in T levels between juvenile and adult males. Overall, our study clearly reflects and confirms the seasonal breeding strategy of this species, showing high similarities to the much better studied European rabbit.

Full text

Please
cite
this
article
in
press
as:
Aguilar,
F.,
et
al.,
Seasonal
changes
in
testosterone
levels
in
wild
Mexican
cottontails
Sylvilagus
cunicularius.
Mammal.
Biol.
(2014),
http://dx.doi.org/10.1016/j.mambio.2014.02.002
ARTICLE IN PRESS
G Model
MAMBIO-40659;
No.
of
Pages
5
Mammalian
Biology
xxx
(2014)
xxx–xxx
Contents
lists
available
at
ScienceDirect
Mammalian
Biology
jou
rn
al
hom
epage:
www.elsevier.com/locate/mambio
Original
Investigation
Seasonal
changes
in
testosterone
levels
in
wild
Mexican
cottontails
Sylvilagus
cunicularius
Fernando
Aguilara,
Heiko
G.
Rödelb,
Jorge
Vázquezc,
Letícia
Nicolasc,
Luisa
Rodríguez-Martínezc,
Amando
Bautistac,
Margarita
Martínez-Gómezc,d,∗
aUniversidad
Autónoma
de
Tlaxcala,
Mexico
bUniversité
Paris
13,
Sorbonne
Paris
Cité,
Laboratoire
d’Ethologie
Expérimentale
et
Comparée
E.A.
4443
(LEEC),
F-93430
Villetaneuse,
France
cCentro
Tlaxcala
de
Biología
de
la
Conducta,
Universidad
Autónoma
de
Tlaxcala,
Mexico
dDepartamento
de
Biología
Celular
y
Fisiología,
Instituto
de
Investigaciones
Biomédicas,
UNAM,
Mexico
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
24
September
2013
Accepted
17
February
2014
Handled
by
Adam
John
Munn.
Available
online
xxx
Keywords:
Hormones
La
Malinche
Net
traps
Reproductive
state
Seasonal
reproduction
a
b
s
t
r
a
c
t
We
studied
serum
testosterone
levels
in
the
endemic
Mexican
cottontail,
Sylvilagus
cunicularius,
which
has
been
reported
to
show
seasonal
breeding.
Animals
were
trapped
in
the
wild
and
in
a
field
enclosure
in
the
National
Park
La
Malinche
in
central
Mexico
over
a
period
of
five
years.
Serum
testosterone
(T)
levels
were
quantified
by
ELISA
from
blood
samples.
T
levels
of
adult
males
were
lowest
around
4
months
after
the
onset
of
the
annual
reproductive
season
and
were
already
high
prior
to
the
onset
of
breeding.
As
expected,
the
T
levels
of
adult
females
were
consistently
lower
than
in
males,
and
there
were
no
differences
in
T
level
with
respect
to
female
reproductive
state.
There
were
no
detectable
sex-specific
differences
in
juveniles
and
subadults,
but
there
was
a
marked
increase
in
T
levels
between
juvenile
and
adult
males.
Overall,
our
study
clearly
reflects
and
confirms
the
seasonal
breeding
strategy
of
this
species,
showing
high
similarities
to
the
much
better
studied
European
rabbit.
©
2014
Deutsche
Gesellschaft
für
Säugetierkunde.
Published
by
Elsevier
GmbH.
All
rights
reserved.
Introduction
Seasonal
breeding
in
mammals
is
a
strategy
for
adapting
to
seasonal
environments,
and
these
changes
in
reproductive
activ-
ity
along
the
season
are
typically
paralleled
by
changes
in
the
underlying
hormonal
status
of
the
animals
(Bronson
and
Heideman,
1984).
Testosterone
(T)
is
the
major
hormone
involved
in
sexual
development,
function
and
behavior
of
mammalian
males
(Nelson,
2005),
and
males
can
show
increased
testosterone
levels
when
females
are
in
estrous
(e.g.
rodents:
Schradin,
2008;
lagomorphs:
Blottner
et
al.,
2000;
ungulates:
Lincoln
and
Kay,
1979;
Sempéré
et
al.,
1992;
Blottner
et
al.,
1996;
Mooring
et
al.,
2004;
primates:
Cavigelli
and
Pereira,
2000).
However,
T
can
also
be
involved
in
female
reproductive
behavior
in
rabbits.
For
example,
T
shows
an
increase
at
days
10–30
of
pregnancy
followed
by
a
significant
decrease
at
day
1
postpartum
in
the
domestic
rabbit
(Oryctolagus
cuniculus:
González-Mariscal
et
al.,
1994).
At
early
gestation
T
levels
contribute
to
digging
behavior,
promote
hair
loosening
(playing
an
∗Corresponding
author
at:
Departamento
de
Biología
Celular
y
Fisiología,
Instituto
de
Investigaciones
Biomédicas,
UNAM,
Mexico.
Tel.:
+52
555
622
6532;
fax:
+52
246
462
1557.
E-mail
addresses:
marmag@biomedicas.unam.mx,
marmagabo@yahoo.com
(M.
Martínez-Gómez).
important
part
in
the
rabbit’s
nest
building
behavior)
and
reduce
food
intake
at
the
end
of
this
period
(González-Mariscal
et
al.,
2003).
Although,
it
seems
that
in
some
seasonal
breeders,
males
become
reproductively
active
before
most
of
females
do.
For
exam-
ple,
male
European
rabbits
(O.
c.
cuniculus)
in
Great
Britain
show
an
increase
in
weight
of
sexual
organs
and
in
the
percentage
of
indi-
viduals
with
active
spermatogenesis
before
the
fecundity
peak
in
females
(Boyd
and
Myhill,
1987).
This
is
confirmed
by
studies
in
the
subspecies
O.
c.
algirus
in
Tunis
and
Portugal,
where
peaks
in
testosterone
level
in
males
were
found
approximately
one
month
prior
to
the
onset
of
the
main
mating
season
(Ben
Saad
and
Baylé,
1985;
Gonc¸
alves
et
al.,
2002).
A
similar
pattern
in
the
changes
of
T
levels
can
be
seen
in
the
European
hare
(Lepus
europaeus:
Blottner
et
al.,
2000).
However,
almost
no
published
information
is
avail-
able
for
the
seasonal
changes
in
T
levels
in
wild
rabbits
of
the
genus
Sylvilagus.
Here,
we
present
data
on
the
endemic
Mexican
cottontail
Sylvi-
lagus
cunicularius.
A
recent
report
on
this
so
far
poorly
studied
species
has
shown
a
clear
pattern
of
seasonal
reproductive
activ-
ity
(Vázquez
et
al.,
2007a).
We
studied
animals
from
the
wild
and
from
field
enclosures
situated
in
the
National
Park
“La
Malinche”
in
Mexico
over
5
years.
Our
main
goal
was
to
(i)
describe
the
sea-
sonal
pattern
of
T
levels
in
males
and
females
and
(ii)
to
investigate
differences
in
the
T
levels
with
respect
to
the
animals’
age
and
reproductive
state.
In
particular,
we
(iii)
intended
to
test
whether
http://dx.doi.org/10.1016/j.mambio.2014.02.002
1616-5047/©
2014
Deutsche
Gesellschaft
für
Säugetierkunde.
Published
by
Elsevier
GmbH.
All
rights
reserved.

Please
cite
this
article
in
press
as:
Aguilar,
F.,
et
al.,
Seasonal
changes
in
testosterone
levels
in
wild
Mexican
cottontails
Sylvilagus
cunicularius.
Mammal.
Biol.
(2014),
http://dx.doi.org/10.1016/j.mambio.2014.02.002
ARTICLE IN PRESS
G Model
MAMBIO-40659;
No.
of
Pages
5
2
F.
Aguilar
et
al.
/
Mammalian
Biology
xxx
(2014)
xxx–xxx
males
of
this
lagomorph
species
also
show
a
distinct
peak
in
T
levels
prior
to
the
onset
of
female
reproductive
activity.
Material
and
methods
Study
species
The
Mexican
cottontail
(S.
cunicularius)
is
one
of
the
largest
species
of
this
genus,
weighing
around
1800–2300
g,
and
it
has
the
widest
distribution
of
all
endemic
cottontails
in
Mexico
(Cervantes
et
al.,
2005).
The
diet
is
mainly
based
on
grasses
(Cervantes
et
al.,
1992;
Hudson
et
al.,
2005).
Mexican
cottontails
breed
all
the
year
around
and
mainly
with
an
increase
in
breeding
from
February
to
October
(Vázquez
et
al.,
2007a).
The
gestation
period
is
34
days
and
females
give
birth
to
litters
of
on
average
3
pups
(range
1–6;
LR,
pers.
obs.).
Study
site
La
Malinche
National
Park
is
located
in
the
central
high
plateau
of
Mexico,
in
the
state
of
Tlaxcala
(19◦1406 N
and
97◦5904 W).
The
climate
in
the
area
is
temperate
semi-humid
with
56%
of
the
annual
rainfall
in
summer
(mean
temperature
9.6 ◦C),
and
less
than
2%
in
winter
(mean
temperature
6.5 ◦C).
The
vegetation
is
a
mix-
ture
of
rough
pasture,
scrub
and
open
woodland
dominated
by
the
trees
(details
in
Sánchez
et
al.,
2005).
Mexican
cottontails
were
rel-
atively
abundant
in
our
study
area
with
about
27
individuals
per
km2(González
et
al.,
2007).
In
addition
to
our
field
study
of
wild
Mexican
cottontails,
we
studied
changes
in
T
levels
in
animals
kept
in
two
field
enclo-
sures.
These
are
located
in
the
Malinche
Research
Station
of
the
Universidad
Autónoma
de
Tlaxcala,
at
3100
m
above
sea
level.
The
enclosures
(530
and
108
m2)
were
surrounded
by
a
wall
(2
m
high)
with
a
wire
mesh
fence
on
top
(2.5
cm
mesh
diameter).
Nylon
cord
strung
at
approximately
20-cm
intervals
across
the
top
of
the
enclo-
sure
prevented
entry
of
aerial
predators.
The
enclosures
contained
wooden
boxes
and
shelters
made
from
native
tussock
grass
(Muh-
lenbergia
macroura)
that
served
as
refuges.
They
also
contained
a
central
concrete
pond
and
food
and
water
troughs.
Each
enclosure
contained
three
to
six
rabbits
of
both
sexes
in
different
age
classes,
all
individually
marked
by
a
color-coded
vinyl
ear
tag
and
with
an
identification
number
tattooed
on
one
ear.
In
addition
to
the
nat-
ural
vegetation
growing
in
the
enclosure,
we
regularly
provided
dried
alfalfa
as
food
source.
The
animals
were
introduced
into
the
enclosures
in
June
2006.
Study
period
and
data
collection
The
study
was
carried
out
from
January
2005
to
April
2009,
when
animals
were
caught
from
the
wild
twice
a
month
using
net
traps
baited
with
alfalfa
(Vázquez
et
al.,
2007b).
Animals
from
the
field
enclosure
were
caught
monthly
since
August
2006.
Once
an
individual
was
captured,
it
was
taken
to
the
research
station.
Ani-
mals
were
weighed
and
blood
was
taken
from
the
medial
ear
vein
(around
1
ml).
Serum
samples
were
then
stored
at
−20 ◦C
until
anal-
ysis.
Before
release,
wild
animals
were
marked
individually
in
the
same
way
as
animals
in
the
enclosures.
Permission
to
live-trap
S.
cunicularius
in
the
National
Park
was
obtained
from
the
Secretaría
del
Medio
Ambiente
y
Recursos
Nat-
urales,
México
(registration
N◦SGPA/dgvs/03502/06).
Hormone
analysis
Serum
testosterone
was
quantified
at
the
Centro
Tlaxcala
de
Biología
de
la
Conducta
using
an
enzyme
immunoassay
(Diagnos-
tic
Automation
Inc.,
Calabasas,
USA).
The
lower
detection
limit
was
0.05
ng/ml.
The
inter-
and
intra-assay
coefficients
of
variation
were
6.3%
and
4.5%
(n
=
6),
respectively.
Data
analysis
and
sample
sizes
Overall,
our
data
set
consisted
of
221
measurements
(128
sam-
ples
from
males
and
93
samples
from
females)
from
a
total
of
82
animals
(46
males,
36
females)
stemming
from
5
different
years.
Note
that
animals
were
frequently
re-trapped
and
thus
were
mea-
sured
repeatedly.
This
was
accounted
for
in
our
analyses
by
the
use
of
mixed-effects
models
(see
below),
allowing
for
the
inclusion
of
repeated
measurements.
99
of
the
measurements
taken
were
col-
lected
from
animals
trapped
in
the
wild
(68
individuals)
and
122
measurements
were
taken
from
animals
living
in
a
field
enclosure
(14
individuals).
We
assigned
three
different
age
classes
based
on
the
animals’
body
mass.
Animals
weighing
less
than
1000
g
were
referred
to
as
“juveniles”,
those
with
a
body
mass
of
1001–1400
g
were
classified
as
“subadults”,
and
animals
with
a
body
mass
of
more
than
1400
g
were
classified
as
“adults”.
In
addition,
maturity
of
males
was
ver-
ified
by
the
presence
of
exterior
testes.
The
reproductive
condition
of
females
was
diagnosed
by
manual
palpation
in
order
to
check
for
pregnancies
and
by
palpation
of
the
mammary
glands
in
order
to
check
for
signs
of
lactation
(Vázquez
et
al.,
2007a).
In
rabbits,
the
annual
onset
of
breeding
can
vary
strongly
among
years
due
to
variation
in
environmental
conditions
(e.g.
O.
cunicu-
lus,
Rödel
et
al.,
2005),
and
such
a
variation
can
be
also
observed
in
the
Mexican
cottontail.
Thus,
we
estimated
the
annual
onset
of
the
breeding
season
based
on
the
first
signs
of
reproductive
activity
of
the
trapped
females,
considering
the
34-day
gestation
and
lactation
period
(Ceballos
and
Galindo,
1984;
LR
pers.
obs.).
This
procedure
revealed
annual
onsets
of
breeding
between
mid
February
and
late
April
(on
average
late
March).
In
order
to
ana-
lyze
changes
in
T-levels
along
the
year,
we
assigned
45-day
time
intervals
with
respect
to
the
estimated
annual
onset
of
the
breeding
season
(see
Fig.
1).
Analyses
were
done
using
the
software
R
version
3.0.0
(R
Core
Team,
2013).
We
calculated
multivariate
linear
mixed-effects
mod-
els
using
the
R
package
lme4
(Bates
and
Maechler,
2010).
We
extracted
the
P-values
using
likelihood-ratio
tests
based
on
changes
in
deviance
(based
on
maximum
likelihood
estimates)
when
each
term
was
dropped
from
the
full
model
including
all
predictor
variables
(Faraway,
2006).
Normality
of
the
model
residuals
was
checked
by
the
Shapiro–Wilk
test
and
visually
by
normal
probabil-
ity
plots.
We
assured
the
homogeneity
of
variances
and
goodness
of
fit
by
plotting
residuals
versus
fitted
values
(Faraway,
2006).
Our
main
goal
was
to
test
for
the
effects
of
different
predictor
variables
on
serum
testosterone
levels.
For
this,
we
included
the
individual
identity
as
a
random
factor
in
order
to
allow
repeated
measurements
of
the
same
animals,
and
we
included
the
assay
identity
in
order
to
statistically
correct
for
potential
between-assay
variation.
We
included
the
origin
of
the
animals
(wild
or
field
enclo-
sures)
as
a
fixed
factor
in
order
to
detect
possible
differences.
Since
this
factor
was
never
significant
(P
>
0.10),
we
concluded
that
pool-
ing
the
two
data
sets
from
animals
stemming
from
the
wild
and
from
the
field
enclosure
did
not
bias
the
results
of
our
study.
Results
Seasonal
changes
in
T
levels
of
adult
males
and
females
In
adult
males,
serum
testosterone
levels
(averaged
over
45-
day
intervals,
see
Fig.
1a)
varied
significantly
during
the
year
(2
5=
32.31,
P
=
0.001).
Post
hoc
comparisons
revealed
a
maximum
peak
in
T
levels
around
90–45
days
prior
to
the
estimated
annual

Please
cite
this
article
in
press
as:
Aguilar,
F.,
et
al.,
Seasonal
changes
in
testosterone
levels
in
wild
Mexican
cottontails
Sylvilagus
cunicularius.
Mammal.
Biol.
(2014),
http://dx.doi.org/10.1016/j.mambio.2014.02.002
ARTICLE IN PRESS
G Model
MAMBIO-40659;
No.
of
Pages
5
F.
Aguilar
et
al.
/
Mammalian
Biology
xxx
(2014)
xxx–xxx
3
Fig.
1.
Serum
testosterone
concentrations
(means
±
SE)
of
adult
male
Mexican
cot-
tontails
measured
during
the
course
of
the
year,
based
on
data
from
5
different
years.
Each
bar
represents
a
45-day
interval
with
respect
to
the
estimated
onset
of
breed-
ing;
sample
sizes
are
given
inside
the
bars.
Differences
among
groups
are
significant
(see
text
for
statistics);
significant
post
hoc
comparisons
after
sequential
Bonferroni
correction
(Holm,
1979)
are
denoted
by
different
letters.
Note
that
the
intervals
“3”,
“4”,
and
“−4”
are
pooled
together
for
analyses
due
to
low
sample
sizes,
and
differ
significantly
from
interval
“−3”.
onset
of
breeding
(statistics
in
Fig.
1a).
There
were
no
differences
among
years
(2
4=
5.20,
P
=
0.27).
We
found
the
same
results
when
using
more
conservative
body
mass
cut-off
values
in
order
to
deter-
mine
adulthood
in
the
trapped
males
(1450
g,
1500
g,
1550
g;
all
P
<
0.01
with
a
similar
maximum
peak
prior
to
the
annual
onset
of
breeding).
In
adult
females,
there
were
no
such
differences
in
T
levels
along
the
year
(2
4=
8.96,
P
=
0.062),
and
also
no
differences
among
years
(2
4=
7.73,
P
=
0.10).
There
were
also
no
significant
differ-
ences
among
females,
which
were
found
to
be
pregnant
(n
=
9),
pregnant
and
lactating
(n
=
16),
or
were
neither
found
to
show
any
signs
of
pregnancy
nor
lactation
(n
=
48;
2
2=
1.80,
P
=
0.41).
On
average,
pregnant
and
pregnant
plus
lactating
females
had
T
levels
of
0.23
ng/ml
(±0.06
SE),
and
non-reproductive
females
had
levels
of
0.14
ng/ml
(±0.09
SE).
Differences
among
age
classes
In
males,
T
levels
differed
significantly
among
the
three
assigned
age
classes
(2
2=
12.86,
P
=
0.002).
Post
hoc
comparisons
revealed
that
adults
had
higher
testosterone
levels
than
juveniles.
But,
there
were
no
significant
differences
between
adults
and
subadults,
or
between
subadults
and
juveniles
(statistics
in
Fig.
2a).
There
were
no
differences
in
T
level
among
age
classes
in
females
(2
2=
1.77,
P
=
0.41;
Fig.
2b).
Sex-specific
comparisons
within
age
classes
revealed
that
such
differences
only
occurred
in
adults
with
males
having
significantly
higher
T
levels
than
females
(2
1=
62.93,
P
<
0.001;
Fig.
2a,b).
On
average,
adult
males
had
T
levels
of
1.01
ng/ml
(±0.11
SE)
and
females
had
levels
of
0.17
ng/ml
(±0.02
SE).
There
were
no
sex-
specific
differences
(all
P
>
0.10)
in
subadults
accounting
on
average
0.53
ng/ml
(±0.11
SE)
in
males
and
0.16
ng/ml
(±0.02
SE)
in
females,
or
in
juveniles
accounting
on
average
0.38
ng/ml
(±0.08
SE)
in
males
and
0.23
ng/ml
(±0.06
SE)
in
females.
Fig.
2.
Comparison
of
testosterone
concentrations
(means
±
SE)
in
(a)
male
and
(b)
female
Mexican
cottontails
of
different
age
classes.
Differences
are
significant
for
males
but
not
for
females,
see
text
for
details
on
statistics.
Different
letters
indi-
cate
significant
post
hoc
comparisons
between
groups
(after
sequential
Bonferroni
correction).
Data
stem
from
5
different
years;
sample
sizes
are
given
inside
the
bars.
Discussion
To
the
best
of
our
knowledge,
his
is
the
first
formal
report
on
serum
testosterone
in
the
genus
Sylvilagus.
T
levels
in
males
ranged
from
0.02
to
4.87
ng/ml
and
this
range
is
close
to
that
of
adult
males
of
the
wild
European
rabbit
(0.06–6.9
ng/ml;
Gonc¸
alves
et
al.,
2002;
Ben
Saad
and
Maurel,
2004).
This
range
is
also
similar
to
levels
of
domestic
(European)
rabbits
(0.26–5.16
ng/ml;
Berger
et
al.,
1982;
Farabollini
1987;
Silván
et
al.,
1990;
Arteaga
et
al.,
2008).
Similarly,
the
T
levels
of
S.
cunicularius
females
were
on
average
0.17
ng/ml
(0.01–1.28
ng/ml)
which
is
close
to
the
range
of
T
levels
published
for
female
domestic
rabbits
(0.20–0.30
ng/ml;
González-Mariscal
et
al.,
1994).
As
expected,
T-levels
were
higher
in
adult
males
compared
to
adult
females.
Typically,
T-levels
in
males
increased
with
age,
in
this
study
rabbits
weighing
more
than
1400
g
had
ele-
vated
T
levels
compared
with
lighter
ones.
For
comparison,
in
male
domestic
rabbits
there
is
a
peak
in
T-levels
of
about
4
ng/ml
around
day
70
of
age
(Berger
et
al.,
1982).
However,
we
did
not
find
any
sex-specific
differences
in
juvenile
or
subadult
Mexican
rabbits
in
serum
T
levels,
as
it
has
been
reported
e.g.,
for
juvenile
domestic
rabbits
with
an
age
of
77
days,
accounting
on
average
2.93
ng/ml
for
males
and
1.98
ng/ml
for
females
(Martiniaková
et
al.,
2008).
Moreover,
there
might
be
sex
differences
in
other
traits
already
apparent
in
juvenile
Mexican
cottontails,
such
as
anatomical
dif-
ferences
in
submandibular
glands
or
contraction
of
airway
smooth
muscle
(Cerbón
et
al.,
1996;
Kouloumenta
et
al.,
2007).
Male
Mexican
cottontails
of
our
study
showed
a
peak
in
serum
T
levels
during
the
winter
(Fig.
1),
well
before
the
estimated
first
con-
ceptions,
based
on
the
timing
of
first
pregnancies
which
we
could
verify
around
mid
March
(Vázquez
et
al.,
2007a).
This
is
similar
to
the
pattern
found
in
European
rabbits
where
T
levels
were
found
to
anticipate
the
main
breeding
season
by
at
least
one
month
(Ben
Saad
and
Baylé,
1985;
Gonc¸
alves
et
al.,
2002;
Ben
Saad
and
Maurel,
2004).
In
European
hares,
although
there
is
a
rise
in
T
during
the
breeding
season,
a
subsequent
increase
of
testosterone
has
been
shown
to
occur
in
autumn
prior
to
the
recrudescence
of
spermato-
genesis
(Blottner
et
al.,
2000).
Further
studies
in
the
Mexican
cottontail
are
necessary
to
explore
possible
changes
in
reproductive
parameters.
For
exam-
ple,
such
studies
might
concern
sperm
count
or
quality,
possible

Please
cite
this
article
in
press
as:
Aguilar,
F.,
et
al.,
Seasonal
changes
in
testosterone
levels
in
wild
Mexican
cottontails
Sylvilagus
cunicularius.
Mammal.
Biol.
(2014),
http://dx.doi.org/10.1016/j.mambio.2014.02.002
ARTICLE IN PRESS
G Model
MAMBIO-40659;
No.
of
Pages
5
4
F.
Aguilar
et
al.
/
Mammalian
Biology
xxx
(2014)
xxx–xxx
changes
in
the
anatomy
or
histology
of
reproductive
organs
and
how
they
are
related
to
changes
in
T
levels.
Another
promising
per-
spective
could
be
the
study
on
associations
between
testosterone
and
territoriality,
aggression
or
dominance
in
S.
cunicularius,
either
in
the
wild
or
under
semi-natural
conditions.
Indeed,
our
studies
in
progress
provide
support
for
territorial
behavior
in
male
Mexican
cottontails
(FA,
HGR,
AB,
LR,
pers.
obs.).
Accordingly,
an
increase
in
aggressive
and
territorial
behavior
of
males
prior
to
the
mating
season
can
be
observed
in
the
European
rabbit
(von
Holst
et
al.,
1999).
For
example,
studies
on
chinning
could
be
conducted
in
the
Malinche
facilities
in
order
to
test
whether
T
levels
are
related
with
the
frequency
of
this
behavior.
Chinning
in
males
needs
low
T
levels
for
showing
up
and
the
frequency
of
this
behavior
has
been
shown
to
be
directly
related
with
territoriality
in
domestic
rabbit
males
(Mykytowycz,
1965;
González-Mariscal
et
al.,
1993).
We
also
investigated
T
levels
in
females
in
order
to
relate
them
with
reproductive
states
and
the
seasonal
reproductive
pattern.
The
T
levels
of
reproductive
females
S.
cunicularius
averaged
0.23
ng/ml
(±0.06
SE),
and
non-reproductive
ones
had
levels
of
0.14
ng/ml
(±0.09
SE)
which,
however,
did
not
differ
statistically.
In
female
domestic
rabbits,
high
T
levels
have
been
reported
to
occur
from
gestation
day
10
(0.26*
ng/ml)
to
day
30
(0.31
ng/ml).
Then
a
sig-
nificant
decline
occurs
at
day
1
of
lactation
(0.20
ng/ml)
and
levels
remain
constant
throughout
the
lactation
period
(0.15–0.20
ng/ml;
*equivalent
in
González-Mariscal
et
al.,
1994).
However,
the
data
collected
in
our
field
study
did
not
allow
us
to
verify
such
a
rela-
tionship
in
S.
cunicularius,
as
it
was
virtually
impossible
to
obtain
a
sufficiently
high
number
of
samples
from
females
in
late
pregnancy.
Future
enclosure
studies
with
more
females
and
with
detailed
mon-
itoring
of
individual
reproductive
status
might
help
to
follow
the
exact
hormonal
patterns
of
female
Mexican
cottontails
along
the
reproductive
cycle.
Investigating
this
pattern
including
changes
of
progesterone,
estradiol
or
prolactin
will
help
to
disentangle
the
physiological
mechanisms
by
a
more
integrative
view
and
enable
comparisons
with
other
lagomorph
species.
In
conclusion,
our
quantification
of
testosterone
levels
in
wild
Mexican
cottontails
underlines
the
seasonal
reproductive
pattern
in
this
species.
Furthermore,
it
provides
another
example
of
a
species,
where
males
show
a
clear
maximum
in
T
levels
prior
to
the
onset
of
the
mating
season.
Acknowledgements
We
are
grateful
to
all
the
students
and
colleagues
who
helped
with
the
field
work
over
the
years,
and
in
particular
we
thank
Minerva
Flores,
Bernardo
Romero
and
Iván
Bravo.
Laura
García
provided
excellent
technical
assistance.
Financial
support
was
pro-
vided
by
the
Posgrado
en
Ciencias
Biológicas,
UAT
and
a
CONACyT
fellowship
provided
to
F.A.
The
study
was
also
supported
by
a
travel
grant
provided
to
A.B.
by
the
Université
Paris
13,
Paris
Sorbonne
Cité,
France.
References
Arteaga,
L.,
Bautista,
A.,
Martínez-Gómez,
M.,
Nicolás,
L.,
Hudson,
R.,
2008.
Scent
marking,
dominance
and
serum
testosterone
levels
in
male
domestic
rabbits.
Physiol.
Behav.
94,
510–515.
Bates,
D.,
Maechler,
M.,
2010.
lme4:
Linear
mixed-effects
models
using
S4
classes.
R
package
version
0.999375-34,
http://CRAN.R-project.org/package=lme4
Ben
Saad,
M.M.,
Baylé,
J.D.,
1985.
Seasonal
changes
in
plasma
testosterone,
thyrox-
ine,
and
cortisol
levels
in
wild
rabbits
(Oryctolagus
cuniculus
algirus)
of
Zembra
Island.
Gen.
Comp.
Endocrinol.
57,
383–388.
Ben
Saad,
M.M.,
Maurel,
D.L.,
2004.
Reciprocal
interaction
between
seasonal
testis
and
thyroid
activity
in
Zembra
Island
wild
rabbits
(Oryctolagus
cuniculus):
effects
of
castration,
thyroidectomy,
temperature
and
photoperiod.
Biol.
Reprod.
70,
1001–1009.
Berger,
M.,
Jean-Faucher,
C.,
de
Turckheim,
M.,
Veyssiere,
G.,
Blanc,
M.R.,
Poirier,
J.C.,
Jean,
C.,
1982.
Testosterone,
luteinizing
hormone
(LH)
and
follicle
stimulating
hormone
(FSH)
in
plasma
of
rabbit
from
birth
to
adulthood.
Correlation
with
sexual
and
behavioural
development.
Acta
Endocrinol.
99,
459–465.
Bronson,
F.M.,
Heideman,
P.D.,
1984.
Seasonal
regulation
of
reproduction
in
mam-
mals.
In:
Knobil,
E.,
Neill,
J.D.
(Eds.),
The
Physiology
of
Reproduction,
vol.
2,
2nd
ed.
Raven
Press,
New
York,
pp.
541–583.
Blottner,
S.,
Faber,
D.,
Roelants,
H.,
2000.
Seasonal
variation
of
testicular
activity
in
European
brown
hare
Lepus
europaeus.
Acta
Theriol.
45,
385–394.
Blottner,
S.,
Hingst,
O.,
Meyer,
H.D.,
1996.
Seasonal
spermatogenesis
and
testos-
terone
production
in
roe
deer
(Capreolus
capreolus).
J.
Reprod.
Fertil.
108,
299–305.
Boyd,
I.L.,
Myhill,
D.G.,
1987.
Seasonal
changes,
reproduction
and
fecundity
in
the
wild
European
rabbit
(Oryctolagus
cuniculus).
J.
Zool.
212,
223–233.
Cavigelli,
S.A.,
Pereira,
M.E.,
2000.
Mating
season
aggression
and
fecal
testos-
terone
levels
in
male
ring-tailed
lemurs
(Lemur
catta).
Horm.
Behav.
37,
246–255.
Ceballos,
G.G.,
Galindo,
L.C.,
1984.
Mamíferos
silvestres
de
la
Cuenca
de
México.
Limusa,
México,
DF.
Cerbón,
M.A.,
Camacho-Arroyo,
I.,
Gamboa-Domínguez,
A.,
González-Mariscal,
G.,
1996.
The
rabbit
submandibular
glands:
sexual
dimorphism,
effects
of
gonadec-
tomy,
and
variations
across
the
female
reproductive
cycle.
J.
Comp.
Physiol.
A
178,
351–357.
Cervantes,
F.A.,
Delgado,
P.,
Colmenares,
A.L.,
2005.
Conejos:
Sylvilagus
cunicularius
(Waterhouse,
1984).
In:
Ceballos,
G.,
Oliva,
G.
(Eds.),
Los
Mamíferos
Silvestres
de
México.
Comisión
Nacional
para
el
Uso
y
Aprovechamiento
de
la
Biodiversidad
y
El
Fondo
de
Cultura
Económica.
Toppan
Printing
Co.,
Hong
Kong,
pp.
842–843.
Cervantes,
F.A.,
Lorenzo,
C.,
Vargas,
J.,
Holmes,
T.,
1992.
Sylvilagus
cunicularius.
Mamm.
Spec.
412,
1–4.
Farabollini,
F.,
1987.
Behavioral
and
endocrine
aspects
of
dominance
and
submission
in
male
rabbits.
Aggress.
Behav.
13,
247–258.
Faraway,
J.,
2006.
Extending
the
Linear
Model
with
R.
Chapman
&
Hall/CRC,
New
York.
González,
J.,
Lara,
C.,
Vázquez,
J.,
Martínez-Gómez,
M.,
2007.
Demography,
density,
and
survival
of
an
endemic
and
near
threatened
cottontail
Sylvilagus
cunicularius
in
central
Mexico.
Acta
Theriol.
52,
299–305.
González-Mariscal,
G.,
Díaz-Sánchez,
V.,
Melo,
A.I.,
Beyer,
C.,
Rosenblatt,
J.S.,
1994.
Maternal
behavior
in
New
Zealand
white
rabbits:
quantification
of
somatic
events,
motor
patterns
and
steroid
plasma
levels.
Physiol.
Behav.
6,
1081–1089.
González-Mariscal,
G.,
Jiménez,
P.,
Beyer,
C.,
Rosenblatt,
J.S.,
2003.
Androgens
stimu-
late
specific
aspects
of
maternal
nest-building
and
reduce
food
intake
in
rabbits.
Horm.
Behav.
43,
312–317.
González-Mariscal,
G.,
Melo,
A.,
Zavala,
A.,
Chirino,
R.,
Beyer,
C.,
1993.
Sex
steroid
reg-
ulation
of
chin-marking
behavior
in
male
New
Zealand
rabbits.
Physiol.
Behav.
54,
1035–1040.
Gonc¸
alves,
H.,
Alves,
P.C.,
Rocha,
A.,
2002.
Seasonal
variation
in
the
reproductive
activity
of
the
wild
rabbit
(Oryctolagus
cuniculus
algirus)
in
a
Mediterranean
ecosystem.
Wildl.
Res.
29,
165–173.
Holm,
S.,
1979.
A
simple
sequential
rejective
multiple
test
procedure.
Scand.
J.
Stat.
6,
65–70.
Hudson,
R.,
Rodríguez-Martínez,
L.,
Distel,
H.,
Cordero,
C.,
Altbäcker,
V.,
Martínez-
Gómez,
M.,
2005.
A
comparison
between
vegetation
and
diet
records
from
the
wet
and
dry
season
in
the
cottontail
rabbit
Sylvilagus
floridanus
at
Ixtacuixtla,
central
Mexico.
Acta
Theriol.
50,
377–389.
Kouloumenta,
V.,
Hatziefthimiou,
A.,
Paraskeva,
E.,
Gourgoulianis,
K.,
Molyvdas,
P.A.,
2007.
Sexual
dimorphism
in
airway
responsiveness
to
sex
hormones
in
rabbits.
Am.
J.
Physiol.
Lung
Cell
Mol.
Physiol.
293,
516.
Lincoln,
G.A.,
Kay,
R.N.,
1979.
Effects
of
season
on
the
secretion
of
LH
and
testos-
terone
in
intact
and
castrated
red
deer
stags
(Cervus
elaphus).
J.
Reprod.
Fertil.
55,
75–80.
Martiniaková,
M.,
Omelka,
R.,
Grosskopf,
B.,
Sirotkin,
A.V.,
Chrenek,
P.,
2008.
Sex-
related
variation
in
compact
bone
microstructure
of
the
femoral
diaphysis
in
juvenile
rabbits.
Acta
Vet.
Scand.
50,
15.
Mooring,
M.S.,
Patton,
M.L.,
Lance,
V.A.,
Hall,
B.M.,
Schaad,
E.W.,
Fortin,
S.S.,
Jella,
J.E.,
McPeak,
K.M.,
2004.
Fecal
androgens
of
bison
bulls
during
the
rut.
Horm.
Behav.
46,
392–398.
Mykytowycz,
R.,
1965.
Further
observations
on
the
territorial
function
and
histology
of
the
submandibular
cutaneous
(chin)
glands
in
the
rabbit,
Oryctolagus
cuniculus
(L.).
Anim.
Behav.
13,
400–408.
Nelson,
R.J.,
2005.
The
endocrine
system.
In:
Nelson,
R.J.
(Ed.),
An
Introduction
to
Behavioral
Endocrinology.
,
3rd
ed.
Sinauer
Associates,
Inc.,
Sunderland,
MA,
USA,
pp.
35–95.
R
Core
Team,
2013.
R:
A
Language
and
Environment
for
Statistical
Computing.
R
Foundation
for
Statistical
Computing,
Vienna,
Austria,
ISBN
3-900051-07-0
http://www.R-project.org
Rödel,
H.G.,
Bora,
A.,
Kaetzke,
P.,
Khaschei,
M.,
Hutzelmeyer,
H.D.,
Zapka,
M.,
von
Holst,
D.,
2005.
Timing
of
breeding
and
reproductive
performance
of
female
European
rabbits
in
response
to
winter
temperature
and
body
mass.
Can.
J.
Zool.
83,
935–942.
Sánchez,
C.,
Windfield,
J.C.,
Fernández,
J.A.,
2005.
Biodiv.
Parque
Nacional
Malinche,
vol.
1.,
pp.
101–137.
Schradin,
C.,
2008.
Seasonal
changes
in
testosterone
and
corticosterone
levels
in
four
social
classes
of
a
desert
dwelling
sociable
rodent.
Horm.
Behav.
53,
573–579.
Sempéré,
J.,
Mauget,
R.,
Bubenik,
G.A.,
1992.
Influence
of
photoperiod
on
the
seasonal
pattern
of
secretion
of
luteinizing
hormone
and
testosterone
and
on
the
antler
cycle
in
roe
deer
(Capreolus
capreolus).
J.
Reprod.
Fertil.
95,
693–700.