Hormones and Behavior 69 (2015) 8–15

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Hormones and Behavior

journal homepage: www.elsevier.com/locate/yhbeh

Regular article

Sexually active males prevent the display of seasonal anestrus

in female goats
J.A. Delgadillo a,⁎, J.A. Flores a, H. Hernández a, P. Poindron b, M. Keller b, G. Fitz-Rodríguez a, G. Duarte a,
J. Vielma a, I.G. Fernández a, P. Chemineau b
a
Centro de Investigación en Reproducción Caprina, Universidad Autónoma Agraria Antonio Narro, Periférico Raúl López Sánchez y Carretera a Santa Fe, C.P. 27054 Torreón, Coahuila, Mexico
b
INRA, CNRS, Univ Tours, Haras Nationaux, UMR 7247 Physiologie de la Reproduction et des Comportements, F-37380 Nouzilly, France

a r t i c l e i n f o a b s t r a c t

Article history: A well-defined season of sexual rest controlled by photoperiod is observed in female sheep and goats during
Received 24 July 2014 spring and summer, delineating their “anestrous season”; bucks also decrease sexual activity at about the same
Revised 24 November 2014 time. Nutrition and/or socio-sexual stimuli play only secondary roles. However, the presence of sexually active
Accepted 2 December 2014
males can reduce the length of seasonal anestrus. Whether it can also completely suppress anestrus has not
Available online 11 December 2014
been investigated. Here we tested this in goats in 3 experiments, using bucks rendered sexually active out of
Keywords:
season by exposure to long days. The continuous presence of these males prevented goats to display seasonal
Caprine anestrus: 12/14 females cycled the year round, vs. 0/13 and 0/11 for females with un-treated bucks or without
Anovulation bucks (experiment 1). When active bucks were removed, females immediately entered anestrus (7/7 stopped
Ovulatory activity ovulating vs. 1/7 if maintained with active bucks; experiment 2). Finally, 7/7 anestrous does with bucks in sexual
Photoperiod rest since 1.5 months commenced cycling rapidly during mid-anestrous, when these bucks became sexually
Sexual behavior active following a treatment with artificial long days, vs. 0/7 with un-treated bucks or no bucks (experiment
Circannual rhythm 3). The presence/withdrawal of active bucks had a highly significant effect in the three experiments
GnRH–LH
(P ≤ 0.002). Therefore, the presence of a mating opportunity can completely override the photoperiodic inhibition
Olfaction
of reproduction of females throughout the anestrous season. Results suggest that we must re-evaluate the relative
contributions of photoperiod vs. other external cues in controlling seasonal reproduction, thus offering new non-
pharmaceutical ways for controlling out-of-season reproduction in small ruminants.
© 2014 Elsevier Inc. All rights reserved.

Introduction
Seasonality of reproduction is a common trait among many wild and
domestic mammals from temperate and subtropical latitudes, allowing
breeding to coincide with optimal availability of food in the natural en-
vironment and protection of young progeny from harsh seasonal condi-
tions (Bronson, 1987, 2009; Helm et al., 2013; Ortavant et al., 1985). In
this respect, female goats and ewes have been used for over 70 years to
investigate seasonality (Bissonnette, 1941; Dardente et al., 2009, 2014;
Hafez, 1952; Malpaux, 2006); they stop ovulating and showing sexual
behavior during a period known as the “anestrous season”, which lasts
from January–February until August–September in the Northern hemi-
sphere (goats: Chemineau et al., 1992a; Duarte et al., 2008, 2010; ewes:
Chanvallon et al., 2011; Karsch et al., 1984; Thimonier and Mauléon,
1969). Males also display a dramatic seasonal decrease in sexual
⁎ Corresponding author at: Centro de Investigación en Reproducción Caprina,
Universidad Autónoma Agraria Antonio Narro, Periférico Raúl López Sánchez y Carretera
a Santa Fe, C.P. 27054, Torreón, Coahuila, Mexico.
E-mail address: joaldesa@yahoo.com (J.A. Delgadillo).
behavior and spermatogenesis at about the same time of the year as
when females are in sexual rest and for the same duration, but with
a one to two-month advance in phase (bucks: Delgadillo et al., 1992,
1999; rams: Lincoln, 1998; Ortavant et al., 1985).
For both sexes and species, changes in photoperiod have been
identified as the main external cue that animals use to synchronize
their circannual endogenous rhythm with the external environment
(Aguilar et al., 2001; Barrell et al., 2000; Duarte et al., 2008; Karsch
et al., 1989). In addition to photoperiod, other natural environmental
cues such as nutrition, temperature or presence of conspecifics may
slightly modify the dates of onset and offset of seasonal reproduction
(Bronson, 2009; Helm et al., 2013; Menassol et al., 2012). Continuous
presence of male goats, for example, has been shown to advance the
onset and to delay the offset of the breeding season of females, leading
to as much as an extra 1.5 months of reproductive activity, compared
with females maintained without males (Restall, 1992). Nonetheless,
the presence of these males, also subjected to the variations of natural
day length, is unable to prevent the seasonal transition to anestrus, a
process considered up till now to be driven exclusively by the annual
changes of photoperiod (Dardente et al., 2014; Helm et al., 2013;
Malpaux, 2006; Restall, 1992).

http://dx.doi.org/10.1016/j.yhbeh.2014.12.001
0018-506X/© 2014 Elsevier Inc. All rights reserved.
 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15
However, males do not need to be continuously present with fe-
males to modify the dates of onset and end of the breeding season.
Males stimulate endocrine and sexual activities in ewes and does
when they are suddenly introduced during the few weeks following
the onset of the anestrous season in females or preceding its end,
whereas their introduction is ineffective in the middle of the anestrous
season. Indeed, the introduction of males activates immediately the
neuroendocrine axis of reproduction in females (Bedos et al., 2014;
Hawken and Martin, 2012; Martin et al., 1986) and leads to synchronous
ovulations within 48 h after contact (Bedos et al., 2010; Chanvallon et al.,
2011; Chemineau et al., 2006; Delgadillo et al., 2009; Martin et al., 1986;
Ungerfeld et al., 2002). This “male effect” has been known for nearly
two centuries and is used in field conditions as a non-pharmaceutical ma-
nipulation to advance reproductive activity by several weeks in anestrous
females (Girard, 1813; Shelton, 1960; Underwood et al., 1944). In addi-
tion, the efficiency of this male effect can be improved by various factors,
in particular the sexual activity of the males. Thus, bucks made sexually
active by previous treatments with artificial light are much more efficient
than those in seasonal sexual rest in inducing ovulation and estrous
behavior in seasonally anovulatory goats, even in the middle of the
anoestrus season (Delgadillo et al., 2002; Flores et al., 2000). This suggests
that the male may play a more important role than usually recognized in
controlling the anestrous season in females. Indeed, taking into account
the increased capacity of light-treated bucks to induce reproductive activ-
ity in anestrous females, the possibility exists that maintaining such males
continuously with females will totally suppress seasonal anestrus.
We tested this hypothesis by monitoring the reproductive activity of
females during three successive years in a seasonal and photoperiod-
sensitive breed of goats using three complementary experimental situ-
ations. In these three experiments, the ovulatory behavior of the female
does was clearly the focus of the study while the males were used as
tools to stimulate female activity. (i) The first experiment determined
whether the long-term and continuous presence of males rendered sex-
ually active by a photoperiod treatment with long days, would prevent
females from becoming seasonally anovulatory and maintain their re-
productive cycles throughout the year and over two consecutive years.
(ii) The second experiment determined whether does that were contin-
uously cycling during the anoestrous season because of the permanent
presence of sexually active males, would cease their reproductive activ-
ity following removal of these photo-stimulated bucks. (iii) The third
experiment determined if the onset of out-of-season sexual activity of
bucks in March, while they were permanently present among does
since 1.5 months and after a period of quiescence of male sexual activity,
was able to rapidly stimulate ovulatory cyclicity during the anoestrous
season of these females. In addition, the results of the second and
third experiments were complementary for the interpretation of the re-
sults of experiment 1 if the hypothesis that active males could overrule
the control of seasonal anestrus by photoperiod proved to be correct. In-
deed, the second experiment would clarify if this effect could be due to
some stimulating effect of cyclic females on anestrous ones, as reported
elsewhere (Restall et al., 1995; Zarco et al., 1995), while the third exper-
iment would allow verifying if the effect could be due to a novelty effect
associated with the swapping between males in experiment 1.
Material and methods
Experimental design and groups
Experiment 1
We used three groups of does (n = 14, 13 and 11). A first group of
does remained in contact with sexually active males for the whole
study and bucks were replaced every two months throughout the
study. During the seasonal anestrus, the males to be used were photo-
stimulated (see details of treatments in next section), so that they
would be sexually active when in contact with does. A second group
of does remained in contact with bucks exposed only to the natural
9
photoperiod, these bucks being sexually active only at the natural
time of breeding, from June–July to December and sexually inactive
from January to June. These un-stimulated bucks were also replaced
every 2 months during the study. In both groups, females were separat-
ed in two sub-groups of 6 or 7 females with 1 male each, to avoid fights
and accidents between males. The contacts between sexes in the two
first groups began on 2 January 2011 and continued until 24 April
2012, when experiment 2 started. A third group of females was totally
separated from bucks (Fig. 1).
We did a daily exchange of the bucks between their respective sub-
groups of does at 09:00 to avoid an individual effect of males. We also
took into account a possible effect of male “novelty” that would be
due to the contact with an unknown male when we changed the
males every 2 months (Hawken and Beard, 2009; Jorre de St Jorre et
al., 2012). To prevent this possibility, we placed the males waiting to
be used in pens adjacent to those of the females about one month before
introducing them; the males were only separated from females by an
openwork wood barrier that allowed visual, olfactory and nose-to-
nose contact between sexes. Vasectomized males were used to avoid
pregnancies.
Experiment 2
This experiment involved the groups of females of experiment 1, and
most importantly those that had males with them, and took place from
25 April to 30 June 2012. A sexually active buck was removed from half
of the females of the sexually active male group of experiment 1 on 25
April 2012. Similarly, an un-stimulated buck was also removed from
half of the females of the sexually inactive male group, thus obtaining
four groups of 6 or 7 females each, two with males, photo-stimulated
or not, and two without males (Fig. 1).
Experiment 3
We used three groups of females (n = 7 each). In the first group,
control does were put in contact with un-stimulated males (n = 2)
from February 1st. In the second group, experimental does were put
in contact with the photo-stimulated, sexually active males (n = 2)
from February 1st. The photo-stimulated bucks were introduced
among does 15 days after the end of their photoperiodic treatment, a
time when they were still sexually quiescent, and about 1.0 month
before the onset of their sexual activity, but when females were still cy-
cling. Introducing the bucks at this time prevented the females from
ovulating in response to a “male effect” and ensured that males would
initiate their sexual activity only after the females had entered anestrus.
In the third group, does were kept away from males throughout the
study (Fig. 2).
In the three experiments, the distance between the various ex-
perimental groups of females was at least 1 km to avoid interference be-
tween them. Males were selected at random within their respective
groups.
Photoperiodic treatments of males
Experiments 1 and 2
We used four groups of 3.5-year-old sexually experienced male
goats: three were stimulated with photoperiodic treatments of long
days and used in the group of females continuously maintained with
sexually active males. The fourth group remained under natural photo-
period and was used in the group of females maintained with males
sexually active during the natural reproductive season and sexually in-
active outside. The three groups of photo-stimulated males (n = 4
males/group, 2 of which were used with the females) consisted of
males rendered sexually active artificially by exposure to photoperiodic
treatments of long days. These males were kept in a shaded open pen
(5 m × 7 m) under natural photoperiodic conditions, but they were ex-
posed to blocks of 2 months of long days, using artificial light in the
morning (06:00 to 08:00) and at night (17:00 to 22:00) to obtain 16 h
10 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15

Exp.2
Experiment 1

2010 2011 2012

S O N D J F M A M J J A S O N D J F M A M J

Subgroups
of light-treated
bucks M M

Does with sexually

active bucks

Control bucks

Does with control

bucks

Does separated
from bucks

Fig. 1. Experimental designs used in experiments 1 and 2 to assess the importance of sexually active male bucks on the maintenance of out-of-season ovulatory activity in female goats. In
experiment 1, three groups of female does were used. One group (n = 14) was permanently in contact during the whole experiment with sexually active bucks which either received
photoperiodic treatments during preceding autumn and winter (3 sub-groups, the last one with implants of melatonin at the end of light treatment), or were used during their natural
breeding season. A second group (n = 13) was in contact with control un-treated bucks which displayed sexual activity from June–July to December. A third group (n = 11) was main-
tained separated from bucks. Sexual activity of males was monitored monthly from January to August of 2011. In experiment 2, which was performed during the last 3 months of exper-
iment 1, bucks were removed from half of the females (May–June 2012). Gray boxes: Exposure to 16 h of light/day in treated bucks; M: melatonin implant in third sub-group of treated
males; the arrow indicates when males are in or out with females. Black boxes: Period of sexual activity of each sub-group of males in experiments 1 and 2. Boxes with stripes: does with
sexually active bucks. Boxes with dots: does with sexually inactive bucks. Open box: does separated from bucks.

of light/day (Chemineau et al., 1992b; Ponce et al., 2014; Zarazaga et al., schedule and light intensity were known to mimic natural long days and
2010). The open pen had 8 daylight lamps of 68 W of energy each. Light- to provoke the same physiological responses as long days in goats
on and light-off were controlled by an electronic timer and light inten- (Chemineau et al., 1992b). The first group of males was treated from
sity was at least 300 lx at the level of the eyes of the animals. These light September 16 to November 15, the second group from November 1 to

Experiment 3

2008 2009
h/day 16

14 Extra light for length

Light light-treated tion of day
l varia
regimen 12 bucks Natura
of bucks 10
N D J F M A M J J A S O
Does with
light-treated
bucks

Does with
control
bucks

Does
separated
from bucks

Fig. 2. Experimental design used in experiment 3. Three groups of female goats (n = 6 or 7 each) were used. One group was in contact with sexually active bucks treated with 2.5 months of
artificially long days (16 h of light/day) during the preceding autumn and winter. These bucks started their induced sexual activity mid-March. A second group was in contact with control
un-treated bucks that displayed sexual activity starting in June and a third group was maintained separated from bucks. Sexual activity of males was monitored in February, March and
May. Gray box: Exposure to 16 h of light/day in treated bucks. The arrows indicate when males were put with females. Empty boxes: does separated from bucks. Boxes with dots: does with
sexually inactive bucks. Boxes with stripes: does with sexually active bucks.
 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15 11

December 31 and the third group from December 15 to February 15.
Bucks were then returned to natural conditions of photoperiod, without
(groups 1 and 2) or with (group 3) two sub-cutaneous implants of mel-
atonin. These photoperiodic treatments induced strong sexual activ-
ity (intense sexual behavior, high plasma concentrations of LH and
testosterone, intense odor of bucks and vocalizations) starting
about 1.5 months after the end of exposure to these long days and
for about 2 months (Delgadillo et al., 2001, 2002, 2012;
Rivas-Muñoz et al., 2007). Therefore, these three groups of bucks
were fully sexually active in January–February, March–April and May–
June respectively and used accordingly in the designated group of females
(Fig. 1).
Un-treated males (n = 14) were kept in a shaded open pen
(6 m × 12 m) like the photoperiod-treated bucks, but they were
exposed only to the natural variation of photoperiod (13 h and
41 min, and 10 h and 19 min of light, at the summer and winter solstices
respectively). These control males were naturally sexually active from
June–July to December and sexually inactive from January to June.
behavior of males (Borja Dos Santos, 2011). Results for this activity are
presented in Fig. 3, showing that photo-stimulated males responded
to the treatments as expected by showing high out-of-season sexual
behavior, while un-treated males were active during the natural breed-
ing season.
General conditions of the study
Description of reproductive characteristics of the breed used and breeding
conditions
The composite breed of goats used in the current studies is from
Northern Mexico, and was the progeny of European populations
imported some centuries ago. This breed has the same reproductive
characteristics as other breeds of goats from temperate and subtropical
latitudes: (i) a strict breeding season, (ii) a circannual endogenous
(a) Sexual activity of males in experiment 1

Experiment 3 200
Two groups of 3.5-year-old sexually experienced male goats were *

Nudging (mean ± SEM)

used. The conditions were similar to those of the males in experiment *
1. The group of photo-induced, sexually active males (n = 4 treated 150
males, 2 of which were used with the females), were exposed to a *
block of 2.5 months of long days (16 h of light/day; from November 1 ns
to January 15), followed by return to natural photoperiod conditions. 100 * *
Males of this group started their sexual activity in mid-March
*
(Delgadillo, 2011; Delgadillo et al., 2002; Ponce et al., 2014). *
The un-treated males of the control group (n = 4, 2 of which were 50
used with the females) were exposed to the natural variation of photope-
riod. These control males were naturally sexually active from May–July to
December.
0
Measurements J F M A M J J A

Females (b) Sexual activity of males in experiment 3

In the three experiments, ovulations were determined by measure-
ment of progesterone (P4) from samples collected once weekly. Concen- 200
trations of plasma progesterone were measured by immunoenzymatic *
Nudging (mean ± SEM)

assay as described by Canépa et al. (2008). Sensitivity was 0.25 ng/mL.

The intra- and inter-assay coefficients of variation were 7 and 3% respec-
tively. Ovulation was considered to have occurred if the level of P4 in a
sample was N 1 ng/mL.
In experiment 1, dates of last and first ovulations for the season were
estimated using these data: date of last ovulation was defined as the
date of the last P4 concentrations higher than 1 ng/mL followed by at
least 2 weeks with P4 concentrations below this value; date of first
ovulation was defined as the date of the first of 2 consecutive concentra-
tions of P4 higher than 1.0 ng/mL, after a series of 3 successive concen-
trations lower than 1 ng/mL. In experiment 3, date of ovulation was
defined as the date of the first of two consecutive concentrations of P4
higher than 1.0 ng/mL.
Males
Sexual behavior displayed by photo-stimulated and un-treated
males was assessed in experiment 1 from January to August 2011 and
in experiment 3 in February, and March for treated males, and February,
March and May for un-treated males; sexual behavior was not assessed
in May in photo-stimulated males because all females of the corre-
sponding group were already cycling (see results). This was done by
regular direct observations in the presence of females by trained ob-
servers who recorded on preformatted sheets the ano-genital sniffing,
nudging, mount attempts, and mounts with intromission (Bedos et al.,
2010, 2014; Flores et al., 2000; Loya-Carrera et al., 2014). However,
only results for nudging were taken into account to verify the presence
of sexual activity in bucks, since it is a reliable variable of sexual
150
100
50
0 NA
J F M A M
Fig. 3. Measurement of sexual activity of bucks in experiments 1 and 3. (a) Experiment 1:
Mean number (±SEM) of nudgings per test in un-treated (□, n = 4) or sexually active
bucks (■, n = 4) during the anestrous season (*P b 0.05, Student t test). Males were ren-
dered sexually active before joining the does by submitting them in open barns to 60 long
days (16 h light/day) followed by natural photoperiod or natural photoperiod and melato-
nin implants. Male sexual activity was determined in 30 min tests, every week from
January to August 2011. (b) Experiment 3: Mean number (±SEM) of nudging per test
in un-treated (□, n = 2) or photo-stimulated bucks (■, n = 2) during the anestrous
season. Males were rendered sexually active by submitting them to 16 h light/day from
1 November to 15 January followed by natural photoperiod, before joining with the
does. Male sexual activity was monitored in 30 min tests, in February and March
in sexually active males, and in February, March and May in un-treated males. Sexual
behavior was not assessed in May in sexually active males (NA) because all females of
the corresponding group were already cycling. Note that in March, only light-treated
bucks were active, while un-treated bucks were active in May.
12 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15
rhythm and photorefractory periods in constant daylength conditions,
and (iii) a high sensitivity to photoperiodic changes for the control of
their breeding activity (Aguilar et al., 2001; Delgadillo et al., 2011;
Duarte et al., 2008, 2010). The males used here for stimulating the fe-
males does were of the same breed and demonstrated the same level
of seasonality (assessed by sexual behavior, spermatogenic activity,
testicular size and odors), as well as the same photosensitivity as the fe-
males (Delgadillo et al., 1999, 2004; Rivas-Muñoz et al., 2007).
Maintenance conditions and group allocation
The experiments were performed in the Laguna region in the State of
Coahuila, Mexico (latitude, 26° 23′N and longitude, 104° 47′W). During
the study, animals had access to alfalfa hay (18% CP) ad libitum and
were provided 200 g per day of a commercial concentrate (14% CP; 2.5
Mcal/kg) per animal, with free access to water and mineral salts. Non-
pregnant multiparous female goats were used in the three experiments.
Well-fed adult goats of good body condition scores were used to avoid
any effect of underfeeding on seasonality. The different groups of females
were balanced for body condition score (BCS: 1 = very lean, 4 = fat;
Walkden-Brown et al., 1997) and allocated to different shaded open
pens (5 m × 5 m), according to their group.
Statistical analyses
The dates (starting from January 1st) of onset and end of ovulatory
activity were compared between groups using the unpaired Student t
test; frequencies were analyzed by the Fisher exact probability test,
with Bonferroni correction when appropriate. All probabilities are
bilateral (Pb).
Ethical note
All procedures used in these experiments were in accordance with
the Guide for the Care and Use of Agricultural Animals in Agricultural
Research and Teaching (Federation of Animal Science Societies. Guide
for the Care and Use of Agricultural Animals in Research, 2010).
Results
Experiment 1
In 2011, 12 of 14 does exposed to the photo-stimulated bucks con-
tinued cycling permanently during the anestrous season; the other
two females stopped cycling for 84 (June–August) and 154 (April–
August) days. In contrast, all 13 control does maintained with un-
stimulated bucks and all 11 does isolated from bucks displayed very
clear-cut periods of anestrus (Fisher exact Pb b 0.001 between the
group in contact with the photo-stimulated bucks versus any of the
two other groups; Fig. 4). The females exposed to un-stimulated
bucks, resumed cycling on 27 July ±9 days, when these bucks had be-
come sexually active (Fig. 3a), while those totally separated from
males resumed ovulation on 17 October ±7 days, showing an anestrus
duration of 142 ± 14 and 242 ± 10 days, respectively (t test = 5.81, P =
0.0001). In 2012, the results were similar: the same 12 of 14 does main-
tained with photo-stimulated bucks continued to cycle until the end of
the experiment on 24 April, whereas does in contact with un-stimulated
males and does without males stopped cycling (Fig. 4).
Experiment 2
On removal of photo-stimulated bucks on 25 April 2012, ovulatory
activity of does immediately ceased after the end of their current luteal
phase, but the females maintained in contact with the photo-stimulated
bucks continued cycling in May and June (0/7 versus 6/7 females cycling
two months after male removal; Fisher exact Pb = 0.005; see right part
of Fig. 4). In contrast, removing un-stimulated males from half of the
a a a a a
100 a a a
a a a a a a a
a
80 a
Goats cycling (%)
60
40
20 b
b b
b b
b b
b b b
0
J F M A M J J A S O N D J F M A M J
2011 2012
Fig. 4. Effects of the continuous presence of the sexually active bucks on ovulatory cyclic
activity of female goats during two consecutive anestrous seasons. Percentage of female
goats cycling per month in does separated from bucks ( , n = 11), maintained with
control un-treated bucks ( , n = 13), or maintained with sexually active males ( ,
n = 14), during two consecutive anestrous seasons (experiment 1). On each month, per-
centages with different superscripts indicate that the corresponding proportions differed
significantly (Fisher exact Pb ≤ 0.001). Active males were removed (gray area) from half
of the females ( , dashed line) during the two last months of the study (experiment
2). Ovulatory cyclicity was monitored by weekly measurements of blood plasma
progesterone.
non-cyclic females that had been with un-treated bucks did not modify
their ovarian activity; all females remained non-cyclic as they had been
during the two previous months.
Experiment 3
The female goats of group 1, which were in contact with the photo-
stimulated bucks, stopped ovulating on 15 February (±5 days), and all
females recommenced ovulating on 17 April (± 3 days) during the
natural anestrous season, soon after the males began to be sexually ac-
tive in mid-March (Fig. 3b). In group 2, in which females were in contact
with un-treated bucks, goats stopped ovulating on 25 February (± 2
days) and started again on 26 July (±6 days). Does of group 3, which
were maintained isolated from males, stopped ovulating on 15 February
(± 4 days) and resumed their ovulatory activity much later, on
26 September (±8 days; Fig. 5). Thus the proportion of cyclic females
in group 1 in April was significantly higher than those in groups 2 and 3
in April, May and June (Fisher exact P b 0.004; Fig. 5).
Discussion
The experimental design that we used in the present experiments
allowed us to disconnect the seasonality of reproduction of male goats
from that of females and to test if seasonal anovulation in does would
remain driven by photoperiod even in the presence of sexually active
bucks. Our results indicate clearly that this was not the case: the
existence of ovulatory activity in female goats was closely linked to
the presence of sexually active males and not to photoperiod. Indeed,
the presence of bucks, if sexually active, maintained ovulatory cyclicity
in does, overriding effectively and completely the seasonal inhibition
of reproduction: almost all females in permanent presence of sexually
active males cycled during the two consecutive anestrous seasons,
while females in presence of control males in sexual rest, stopped
cycling each year for about 5 months (experiment 1). Therefore, it ap-
pears that in the female goat, the sexual activity of the male is at least
as important as photoperiod in controlling the seasonal rhythm of ovu-
latory cyclicity. In other words, the seasonal control of reproduction by
 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15
a a
100
80
a of anestrus is caused by the sexual activity of the males and not by some
Goats cycling (%)
60
40
20 b as temperature, food and socio-sexual interactions were supposed to
b b
0 2009; Helm et al., 2013; Ortavant et al., 1985). Clearly, this needs to be
J F M A M J J A S O
Fig. 5. Experiment 3: Effects of sexual activity of photo-stimulated males on the ovulatory
activity of female goats during seasonal anestrus. Percentage of female goats cycling per
month in 3 groups of does (n = 7 in all groups) maintained either separated from bucks
( ), kept with control un-treated bucks ( ), or kept with the sexually active males
( ) during the anestrous season. On each month, percentages with different super-
scripts indicate that the corresponding proportions differed significantly (Fisher exact
Pb ≤ 0.02). Ovulatory cyclicity was determined by weekly measurements of blood plasma
progesterone.
photoperiod in the female goat may be more indirect than previously
thought; photoperiod appears to act primarily by controlling sea-
sonality of sexual activity in the male, which in turn drives that of
the female. Nonetheless, this is not to say that photoperiod plays
no role in females, since they show a clear-cut seasonal period of an-
estrus when no bucks are present and the display of this anestrus
can be modified by photoperiod manipulations (Chemineau et al.,
1992b; Duarte et al., 2010).
Results of experiment 2 confirm that the failure of females to display
seasonal anestrus did not depend on some entraining effect of a few cy-
clic females on the rest of the group, even though such a female–female
effect has been reported in other conditions (Restall et al., 1995; Zarco
et al., 1995). In fact, the effect of cyclic goats to induce reproductive activ-
ity in females is not as clear-cut and reliable as the male effect (Véliz et al.,
2002), and it cannot explain our present results. Here, females stopped
cycling immediately after the sexually active males were withdrawn
from the group, even though females had been cycling continuously for
one year and a half. In case of a female–female effect, some cyclic activity
would have continued and the termination of cyclicity would not have
taken place or would have been at least more progressive. Therefore, in
the present experiments, the maintenance of cyclicity in females during
the natural anestrous season cannot be explained by some socio-sexual
interactions between females but depended on the presence of sexually
active bucks in the group, as in the study of Véliz et al. (2002).
Generally, the activation of reproductive activity in anestrous fe-
males by introducing a male in the group combines at least two factors:
the novelty of the stimulus and its sexual activity. In fact, in sheep the
lack of familiarity of the stimulus male has long been considered to be
an important factor for a good response in the females (Hawken and
Beard, 2009; Jorre de St Jorre et al., 2012), even though several studies
in goats have now shown that the level of sexual activity of the male
plays a critical role (Delgadillo et al., 2006; Martínez-Alfaro et al.,
2014; Vielma et al., 2009). The results of experiment 3 confirm without
any ambiguity, at least in the goat, that it is the sexual activity of the
male and not its novelty that induces the ovarian response of the fe-
males. Indeed, the photo-stimulated males had been present for more
than a month with the females, but these reinitiated ovarian activity
only when the males became sexually active. Similarly, females kept
with un-stimulated males started their ovulatory activity once the
13
males became active in June, whereas females kept separated from
males started ovulations significantly later.
Therefore, as a whole our results confirm that the onset and mainte-
nance of reproductive activity of female goats during the natural season
other social changes in the group and that this was also the case in ex-
periment 1. This raises the question of the relative importance of the dif-
ferent environmental cues which drive seasonal breeding in this species
and breed. So far, photoperiod was considered as being the main cue
which entrains the putative circannual reproductive rhythm to cope
with the available environmental resources and enhances reproductive
success and perpetuation of such seasonal species. The other cues, such
play minor roles compared to changes in day length (Bronson, 1987,
re-evaluated.
Regarding the physiological mechanisms involved in the effect
described here, we already know a large part of the neuroendocrine
mechanisms of seasonality which have been extensively explored in
sheep and goats as well as in many other species (Dardente et al.,
2014; Malpaux, 2006; Nakao et al., 2008). These studies clearly indicat-
ed that the central regulation of GnRH/LH pulsatility is the key element
of seasonality of reproduction in sheep, a dramatic decrease of its
pulsatility in late winter-early spring leading to the onset of anestrus
(Barrell et al., 1992; Hawken and Martin, 2012; Karsch et al., 1984).
We also know that, in sheep and goats, sexually active males are espe-
cially active to stimulate GnRH and LH pulsatility immediately after in-
troduction of the male or its odor during the anestrous season (Bedos
et al., 2014; Murata et al., 2014; Vielma et al., 2009).
Thus, it is very likely that in the present experiments, and especially
in the first one, the permanent presence of sexually active bucks was
able to reactivate the GnRH/LH pulsatility in does after luteolysis,
preventing the cessation of cycling. This would be also the case in exper-
iment 3 in which males progressively increased their sexual activity
mid-March, stimulating GnRH/LH pulsatility in the females and provok-
ing the onset of out-of-season breeding activity. Conversely, in ex-
periment 2, it is most likely that GnRH/LH pulsatility was no longer
sustained after the end of the luteal phase, once active males were with-
drawn, in spite of the presence of other cycling does in the group, pro-
voking an immediate cessation of the ovulatory cycles. Thus, in the
present experiments, the stimulation of pulsatile activity of GnRH/LH
by the sexually active males appears the most likely process by which
males prevented the seasonal inhibition of this pulsatility, which nor-
mally occurs at the end of winter or beginning of spring in presence of
sexually inactive males or in their absence.
To our knowledge, this study is the first to demonstrate that sexual
activity of the male can override completely the photoperiodic control
of seasonality in females of a species with a circannual rhythm of repro-
duction. The extent to which these results can be extended to other
breeds of goats and under other latitudes remains open to discussion.
The breed of goats that we used in this study has all the characteristics
of a seasonal and photoperiod-sensitive breed. It is a mixed breed
resulting from crosses between various seasonal European breeds. It
displays marked breeding seasons of about 6 month duration, with a
circannual reproductive endogenous rhythm independent of food avail-
ability (Aguilar et al., 2001; Duarte et al., 2008), as reported in others
breeds of sheep and goats from temperate latitudes (Gómez-Brunet
et al., 2008; Karsch et al., 1989). Furthermore, it shows “classical” repro-
ductive responses to short days and to long days, as well as existence of
periods of refractoriness to long days and to short days (Delgadillo et al.,
2011; Duarte et al., 2010), as in Saanen dairy goats (Gebbie et al., 1999).
Thus, one could expect that other seasonal breeds of goats will respond
in the same way as the breed used in the present study, even though
further studies are needed to confirm this point.
Another point to consider is the possibility that the response of the
females we obtained here depended on the subtropical latitude under
14 J.A. Delgadillo et al. / Hormones and Behavior 69 (2015) 8–15
which our study was carried out. We may wonder if the moderate var-
iation of day length existing throughout the year, from about 10 h in
winter to 14 h per day in summer, could be responsible for unmasking
the effect of non-photic environmental cues as reported in the hamster
(Paul et al., 2009a, 2009b). Here again, more studies are needed to clar-
ify this point. Nonetheless, it must be pointed out that so far all studies
carried out under the latitude of the present study have produced re-
sults similar to those obtained under temperate latitudes and it may
be that the effect reported here was never revealed before because it
was not tested previously.
It is well established that the neurobiological control of seasonal
breeding is similar in goats and sheep. Therefore, it would be reasonable
to expect that the results obtained here will apply also in sheep, as sug-
gested by some recent results obtained in Spain (Abecia Alfonso, per-
sonal communication). On the other hand, it must be underlined that
in various breeds of sheep, rams tend to maintain some sexual activity
even during the natural period of seasonal rest, contrary to goat bucks
which show a more complete inhibition of their sexual behavior at the
time females are in seasonal anoestrus. Therefore, it cannot be excluded
that in sheep, social novelty of the stimulus male plays a more impor-
tant role than in goats, at least in those breeds whose males maintain
some sexual activity all year round (Hawken and Beard, 2009; Jorre de
St Jorre et al., 2012). Here again, further studies are needed to investi-
gate to what extent the present results can apply to sheep, but also to
other seasonal ruminants or any species in which seasonal reproduction
is controlled by photoperiod. For example, this could be the case in hors-
es, since exposure to stallions advances the onset of the breeding season
of mares (Wespi et al., 2014).
Conclusion
Our results show that female goats can bypass the seasonal inhibi-
tion of reproduction if they have the opportunity to mate with a sexual-
ly active male. This capacity of the male to override the control of female
sexual behavior by photoperiod generates new questions for research
not only in goats and sheep, but also in all species in which photoperiod
is recognized as the main factor controlling seasonality of reproduction.
Our results plead for a re-equilibrium in favor of non-photic environmen-
tal cues, especially socio-sexual cues, which were not really explored in
details until now. Finally, they open new and non-pharmaceutical ways
for controlling out-of-season reproduction in agricultural species.
Conflict of interest statement
The authors declare no competing financial interests. Preliminary
results of experiment 3 were published as an abstract form in the 44th
ISAE Conference in Uppsala, Sweden, 3–7 August 2010.
Acknowledgments
We wish to thank all members of the Centro de Investigación en
Reproducción Caprina (CIRCA) for their participation in the experi-
ments. We also thank Anne-Lyse Laisné and the Assay Laboratory in
INRA-Nouzilly for doing the progesterone assays. We are grateful to
Benoît Malpaux for his help in designing the experiments, David R. Lind-
say, Lance Kriegsfeld, Jacques Balthazart and Donald Skinner for valu-
able discussion and/or feedback on the manuscript. We thank Alfonso
Abecia for his helpful income in the discussion about the same experi-
ments in sheep. Dolores López Magaña helped with the figures. Part of
this research was supported by a joint ANR-CONACYT grant to J.A.D. and
M.K. in Mexico and France (159884): El efecto macho en ungulados:
reproducción aplicada y mecanismos neuroendocrinos (11-ISV7-001-
01), and by the Research Direction and Postgraduate Direction from the
Universidad Autónoma Agraria Antonio Narro.
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