Reprod Dom Anim 49, 977–984 (2014); doi: 10.1111/rda.
12417
ISSN 0936–6768
Fertility of a High-Altitude Sheep Model is Compromised by Deficiencies in Both
Preovulatory Follicle Development and Plasma LH Availability
VH Parraguez1,2,3, F Diaz1, E Cofr
e1, B Urquieta1, M De Los Reyes1, S Astiz4 and A Gonzalez-Bulnes4
1
Faculty of Veterinary Sciences, University of Chile, La Granja, Santiago, Chile; 2Faculty of Agricultural Sciences, University of Chile, La Granja,
Santiago, Chile; 3International Centre for Andean Studies, University of Chile, La Granja, Santiago, Chile; 4Comparative Physiology Lab, SGIT-
INIA, Madrid, Spain
Contents
At high altitude, hypoxia and/or oxidative stress may com-
promise fertility. This study tested the relative effect of short-
or long-term exposure to high-altitude hypobaric hypoxia and
oxidative stress in sheep on preovulatory follicle dynamics and
gonadotrophin secretion. Thus, growth dynamics, stereido-
genic function and competence to ovulate of preovulatory
follicles, as well as FSH and LH availability throughout the
entire oestrous cycle, were compared among sheep native from
low and high altitude, and sheep newcomers to high altitude.
The results indicates that short-term exposure in sheep
newcomers to high altitude has a deleterious effect on both
the ovarian function (affecting preovulatory follicular devel-
opment) and the pituitary function (diminishing plasma LH
availability). On the other hand, there were no detected
differences in the preovulatory follicular development in sheep
adapted to high altitude for generations and, conversely, LH
secretion was increased, which suggests an adaptive mecha-
nism. The treatment with antioxidant agents during a relative
short period for the time of folliculogenesis (approximately
1 month and a half) changed substantially the development of
preovulatory follicles in short-term exposed sheep to similar
patterns than in sheep native and living to both high and low
altitude. These results highlight the role of oxidative stress in
the detriment of the reproductive function in individuals
recently exposed to high-altitude hypoxic environment.
Introduction
Fertility of females at high-altitude environments is
reduced when compared to low-altitude counterparts,
both in humans (Vitzthum et al. 2000) and in domestic
animal species (mainly sheep) introduced to high
plateaus (Lanino 1977; De Carolis 1987; Parraguez
et al. 2005, 2006). The low reproductive efficiency is
even more marked in newcomers to high altitude, by
acute exposition to hypoxia, both humans (Moore et al.
2001, 2004; Hartinger et al. 2006) and sheep (Parraguez
et al. 2005, 2006). These facts are of main importance,
firstly, for human medicine; approximately 140 millions
of people inhabit at altitudes above 2500 m above sea
level (m.a.s.l.), and approximately 40 millions of people
visit regions above 2500 m.a.s.l. every year (Moore et al.
1998). Second, sheep is a main economical resource for
approximately 25 millions of rural people established at
altitudes above 2500 m.a.s.l. in developing regions and
transition countries, like the Andean and Qinghai-
Tibetan high plateaus (Huddleston et al. 2003).
Most of the studies about exposition to high-altitude
hypoxia have been focused on its effects during preg-
nancy, resulting in embryo/foetal losses, intrauterine
growth restriction and reduced birth weight both in
© 2014 Blackwell Verlag GmbH
humans (Jensen and Moore 1997; Moore et al. 2001)
and in animals (Parraguez et al. 2005, 2006). However,
fertility is also conditioned by fecundity (capacity for
conceiving) and the studies on fecundity and, specifi-
cally, on ovarian activity (including direct observation
of folliculogenesis, ovulation and luteal function) are
scarce. The available data on ovarian activity in human
beings are only based on interviews and evaluation of
steroids in saliva or urine, mainly for establishing the
duration and regularity of the menstrual cycle (Vitzthum
et al. 2000; Vitzthum 2001), and the data obtained are
usually biased by concurrent factors (economic impov-
erishment, malnutrition and behavioural and sociocul-
tural factors; Vitzthum and Wiley 2003). In sheep, there
is also scarce information. A previous study, determin-
ing the occurrence of oestrus and ovulation in ewes that
were native and na€ıve to high altitude, showed that 82%
of high-altitude native ewes but only 32% of the
newcomers were in oestrus, and finally, 48% of the
native sheep but none in the newcomers group become
pregnant (Parraguez et al. 2006). These outcomes con-
trast with the data existing for sheep at sea level and
suggest deficiencies in the ovarian features that, besides
embryo/foetal losses, impair fertility.
Such hypothesis is supported by a recent study in which
exposure of sheep to high-altitude hypobaric hypoxia,
both during short and long time, negatively affected
corpus luteum development and function (Parraguez
et al. 2013). The quality of corpora lutea has been
classically linked to both ovarian causes (quality of the
previous preovulatory follicle; Keisler and Keisler 1989)
and/or systemic causes (inadequate LH secretion, which
is necessary for the final maturation of the preovulatory
follicles and, subsequently, adequate development of the
corpora lutea; Adams 1999; Campbell et al. 1999). Thus,
it becomes evident the necessity of increasing the infor-
mation about the effects of the exposition to high altitude
on such features; in consequence, the present experiment
aims to characterize growth dynamics, stereidogenic
function and competence to ovulate of preovulatory
follicles, as well as to determine plasma FSH and LH
concentrations throughout the entire oestrous cycle. As
alterations in fertility at high altitude seem to be related to
processes of oxidative stress and, thus, fertility might be
favoured by antioxidant therapies (Parraguez et al.
2006), a concomitant objective of the study was to assess
the effects of the daily administration of antioxidant
vitamins C and E on preovulatory follicle development
and function (in terms of oestradiol secretion), and
gonadotrophins (FSH and LH) secretion.
978 VH Parraguez, F Diaz, E Cofr
e, B Urquieta, M De Los Reyes, S Astiz and A Gonzalez-Bulnes

Materials and Methods The other 24 ewes were native to a low-altitude

This study was performed, at the International Center
for Andean Studies (INCAS, University of Chile, Putre,
Chile; 3589 m.a.s.l.), in accordance with the Interna-
tional Guiding Principles for Biomedical Research
Involving Animals (Council for International Organisa-
tion of Medical Sciences, World Health Organization)
and was approved by the Bioethics Review Committee
of the Faculty of Veterinary Sciences, University of
Chile, as well as by the Bioethics Committee of Chile’s
National Agency for Scientific Research (Comisi
on
Nacional de Investigaci
on Cient
ıfica y Tecnol
ogica,
CONICYT). A summary of the experimental design is
presented in Table 1.
Animals and experimental procedure
The experiments, conducted during the breeding season
(April–May), involved a total of 36 Creole ewes
(Chilean mix breed descendants from Churra and
Manchega Spanish breeds) with an average body weight
of 40.1  3.4 kg, adequate body condition (2.6  0.2
on a scale of 1–5) and having a history of, at least, two
previous pregnancies and normal parturitions. Twelve
of the animals were native to high altitude (3500–4000
m.a.s.l.; barometric pressure approximately 660 hPa;
group HH). These animals were descendants of the
sheep herds introduced to the plateau by the Spanish
settlers and have thus been adapted to hypobaric
hypoxia for over 500 generations.
condition (~500 m.a.s.l.; barometric pressure approxi-
mately 990 hPa) and selected on the basis of similar
phenotypes, body weights and age as the females in the
HH group. Half of these animals (group LH, n = 12)
were moved to the INCAS facilities for joining the HH
ewes. The remaining 12 ewes were maintained at a low
altitude (group LL). Animals were provided with alfalfa
hay daily (~2 kg/day; dry matter = 89.8%, metaboliz-
able energy = 10.6 MJ/kg, crude protein = 14.0%) and
fresh water ad libitum. The food supply was calculated
for fulfilling daily requirements (NRC 1985).
To determine the effects of antioxidant treatments,
six ewes from each group were randomly selected for
receiving antioxidant vitamin supplementation (groups
HHV, LLV and LHV), administered daily from Day
30 prior starting the study (at the oestrous cycle
following to a first oestrus synchronized with clopros-
tenol) to the end of the experiment. In the treated
groups, 0.3 kg alfalfa was supplemented with 500 mg
of vitamin C and 350 IU of vitamin E per animal and
administered in individual feeding, early each morning.
These doses and administration way have demon-
strated to significantly increase plasma concentrations
of the corresponding vitamins and to reduce the
oxidative damage in ewes at high altitude (Parraguez
et al. 2011). After the consumption of this ration, the
remaining amount of alfalfa was given to the ewes for
the completion of their daily requirements (NRC
1985).

Table 1. Summary of the experimental design

Experimental day Experimental procedure

0  Transfer of low-altitude native sheep to a high-altitude experimental station and formation of

the experimental groups:

— HH; ewes from high altitude maintained at high altitude; n = 6

— HHV: ewes from high altitude maintained at high altitude and treated with vitamins; n = 6
— LH: ewes from low altitude moved to high altitude; n = 6
— LHV: ewes from low altitude moved to high altitude and treated with vitamins; n = 6
— LL: ewes from low altitude maintained at low altitude; n = 6
— LLV: ewes from low altitude maintained at low altitude and treated with vitamins; n = 6

 Start treatment with vitamins C and E in groups HHV, LHV and LL

 Surgery for installation of the arterial and venous catheters
 Administration of the first dose of cloprostenol for oestrus synchronization
9  Administration of the second dose of cloprostenol for oestrous synchronization
 Introduction of vasectomized males for detection of females in oestrus
11–12  First oestrus after synchronization treatment
24–27  Daily sampling of venous blood for the measurement of FSH and LH
27–30  Second oestrus after synchronization treatment
 Sampling of arterial blood for evaluation of oxygenation status
39–47  Daily ultrasound examinations for evaluation of preovulatory follicle development
 Daily sampling of venous blood for the measurement of oestradiol
45–47  Third oestrus after synchronization treatment
55  Assessment of ovulation rate

In the representation of the experimental groups, the first letter of the acronym is the altitudinal origin and the second letter is the altitude where the animals stayed
during the study (H for high altitude, L for low altitude). If the animal was supplemented with vitamins, the letter V was added. Therefore, there are three non-
supplemented groups (HH, LH and LL) and three vitamin-supplemented groups (HHV, LHV and LLV).

© 2014 Blackwell Verlag GmbH

Preovulatory Follicle Development at High-Altitude
At the same day in which vitamins supplementation
started, polyvinyl catheters (2.5 mm internal diameter,
Tygonâ, Saint Gobain Performance Plastics, Akron,
OH, USA) were surgically inserted into the blood
vessels for hormones, gases and oxidative stress evalu-
ation, avoiding consecutive vessels puncture. All the
ewes were anesthetized (ketamine clorhidrate 20 mg/kg
i.m.; Ketamilâ, Troy Laboratories, Smithfield, Austra-
lia), and catheters were placed into the left femoral
artery and vein. Once installed, they were filled with
heparinized saline (1000 IU/ml) to prevent clotting and
were then passed through the subcutaneous tissue to the
left flank of the ewe, where they were exteriorized and
placed in a canvas pocket attached to the skin.
Oestrous cycles were synchronized by the administra-
tion of two i.m. doses of 125 lg cloprostenol (Ovoluteâ,
Drag Pharma, Santiago, Chile), 9 days apart. Oestrous
detection was performed with trained vasectomized
rams, daily from the day after the second cloprostenol
administration until the detection of oestrous signs. The
study of both ovarian and pituitary function was
performed during the second oestrous cycle after clopr-
ostenol treatment.
Arterial blood samples (1 ml), collected at the begin-
ning of the second oestrus, were used for the assessment
of the oxygenation status of the animals. Venous blood
samples (5 ml), daily collected from approximately
3 days before the estimated onset of the second oestrus
and during the second oestrous cycle, were used for
assessment of plasma concentrations of the hormones
FSH and LH. These samples were obtained in heparin-
ized syringes, using the intravenous catheters, and
centrifuged at 800 9 g 9 5 min. Plasma aliquots were
recovered and stored at 20°C for measurement of
oestradiol, FSH and LH.
Assessment of oxygenation status
Oxygenation status was assessed by determining the
partial pressure of oxygen (PaO2), partial pressure of
carbon dioxide (PaCO2), haematocrit value (Ht), haemo-
globin concentration (Hb), Hb saturation by oxygen
(SatHb) and pH value. These measurements were per-
formed in a IL Synthesis 25TM gas analyzer (Instrumenta-
tion Laboratory, Lexington, MA, USA), calibrated to a
local atmospheric pressure and ovine body temperature.
Assessment of preovulatory follicular development
Patterns of growth of the preovulatory follicles were
assessed daily by transrectal ultrasonography for deter-
mining their largest diameter and development in
successive observations. Observations of the follicles
were retrospectively classified from Day -5 to Day 0
when onset of the oestrus following the studied cycle
was determined and considered as Day 0. The ultrason-
ographies were performed with an Aloka SSD500
(Aloka Co. Ltd., Tokyo, Japan) equipped with a
7.5 MHz linear-array transducer (Aloka UST 600-7.5).
Scanning was performed as previously described by
Schrick et al. (1993) and validated by comparison with
histological findings (Gonzalez-Bulnes et al. 1994). In
brief, observations were conducted with the sheep
© 2014 Blackwell Verlag GmbH
979
placed in dorsal recumbence on a metallic cradle. After
introducing a hydrosoluble contact gel into the rectum
to enhance ultrasound transmission, the probe was
rotated clockwise and counterclockwise to observe both
ovaries and their structures and to obtain the largest
diameter of the largest follicle, which was measured by
means of the internal callipers of the ultrasound
machine.
Assessment of plasma concentration of oestradiol, FSH
and LH
Plasma concentration of LH and FSH was measured in
200 ll plasma by specific RIAs using reactive and
instructions provided by the National Institute of
Diabetes and Digestive and Kidney Diseases (NIDDK,
NIH, USA), and validated for the ovine species by
Recabarren et al. (1996). Sensitivity of the assays was
0.2 ng/ml, whilst the intra- and interassay variation
coefficients were 5.0% and 12.0%, respectively, for LH,
and 6.0% and 10.0%, respectively, for FSH.
Plasma concentrations of 17b-oestradiol were deter-
mined only in the samples obtained between days -5 and
0, concomitantly with ultrasound scanning. Concentra-
tion of 17b-oestradiol was measured in 100 ll plasma by
a double antibody RIA without prior extraction, using
reagents and techniques provided by the Spectria
radioimmunoassay kit (Orion Diagnostic Corp, Espoo,
Finland), as described by Romeu et al. (1995) and
adapted for ovine plasma (Gonzalez-Bulnes et al. 2003).
Sensitivity of the assay was 0.5 pg/ml, whilst the intra-
and interassay variation coefficients were 3.5% and
6.1%, respectively.
Assessment of ovulation rate
In all the animals, number of corpora lutea was
determined by ultrasonography, using the Aloka
SSD500 and the 7.5 MHz linear-array transducer as
previously described, between 8 and 10 days after the
studied follicular phase.
Statistical analysis
The experimental data were compared by analysis of
variance using the general linear model procedure
(GLM; SAS Institute Inc., Cary, NC, USA), with
preovulatory follicular development and changes in
blood concentrations of estradiol, FSH and LH being
compared by analysis of variance for repeated measures
(split-plot ANOVA). Comparisons were made using two
statistical models. The first model was used to test the
effect of altitude, including the two following cross-
factors: the place of birth or altitudinal origin of the
animals and the place where the reproductive cycles
were studied (high or low altitude). The second model
took into consideration the previous two factors in
addition to the antioxidant vitamin supplementation
(yes or not). When significant differences were found,
Duncan’s test was used to determine the groups among
which the differences were statistically significant. In
addition, growth of the dominant follicles as a function
of the time was evaluated during the last 5 days before
980 VH Parraguez, F Diaz, E Cofr
e, B Urquieta, M De Los Reyes, S Astiz and A Gonzalez-Bulnes

ovulation by means of Pearson’s correlation. A prob-
ability of p < 0.05 was considered statistically signifi-
cant. The results are expressed as the means  SEM.
Results
Influence of altitudinal origin on oxygenation status and
effects from antioxidant treatments
The assessment of arterial blood gases and other
variables related to oxygen transport (Table 2) showed
an evident hypoxaemia state in the high-altitude groups
(groups HH and LH) when compared to low-altitude
groups (LL). The treatment with antioxidants did not
affect significantly these variables in the group LLV, but
modified PaCO2 and Ht in sheep in both groups LHV
group LHV. In this group, the pattern of growth of
preovulatory follicles was clearly modified by antioxi-
dants, being different from those in group LH and similar
to those of the other groups. The size of the ovulatory
follicle in the group LHV increased with time (r = 0.862,
p < 0.00005) and its mean size was significantly smaller
(p < 0.05) than in the group LH from Days -5 to -3 of
oestrous detection (Day 0), similar at Days -2 and -1 and
larger at Day 0 (p < 0.05; Day 0 = day of onset of the
oestrus following the studied cycle).
Assessment of the plasma 17b-oestradiol levels
(Fig. 1b) showed no significant differences among
(a) 8
c HH

Follicular diameter (mm)

and HHV and Hb content in HHV. b HHV
6
a LH
LHV
Effects of altitudinal status and antioxidant treatment on 4
preovulatory follicular development LL
LLV
The assessment of the number of corpora lutea showed
2
no significant differences in the mean ovulation rate
between sheep in the groups LL, HH and LH
(1.4  0.5, 1.3  0.3 and 1.4  0.2 corpora lutea, 0
respectively). However, there were significant differences –5 –4 –3 –2 –1 0
in the size and developmental patterns of the ovulatory Days relative to ovulation
(b)
follicles during the follicular phase (Fig. 1a). At the
30
Plasma 17β-estradiol (pg/ml)

beginning of the follicular phase, the preovulatory HH

follicles were significantly larger in the sheep of the * HHV
group LH than in the groups LL and HH (p < 0.05). LH
Afterwards, the size of the preovulatory follicles in the 20
LHV
group LH remained almost in the same size during the
follicular phase (r = 0.410, n.s.), whilst preovulatory LL
follicles grew significantly over time in groups LL and 10 LLV
HH (r = 0.984, p < 0.001 and r = 0.861, p < 0.0001,
respectively). At oestrous onset, preovulatory follicles
were finally smaller in the sheep both the groups LH and 0
HH than in the group LL (p < 0.05). –5 –4 –3 –2 –1 0
The administration of antioxidant supplementation Days relative to oestrus
showed no significant effects on the mean ovulation rate Fig. 1. Follicular growth (a) and plasma 17-b oestradiol (b) during the
of sheep either in the groups LLV (1.3  0.5 corpora last 5 days before oestrous onset in cycling sheep at a high altitude.
lutea), LHV (1.5  0.3 corpora lutea) or HHV (1.5  0.3 Day 0 = day of onset of the oestrus following the studied cycle. In the
corpora lutea); the increase in the ovulation rate of left panel, letter ‘a’ indicates that the group LH is different from all
vitamin-treated sheep did not reach statistical significance other groups; letter ‘b’ indicates that group LH is different only from
groups LHV, LL and HHV; letter ‘c’ indicates significant differences
when compared to counterpart groups without treat- between groups kept at high and low altitude. In the right panel,
ment. The administration of antioxidants showed effects asterisks indicate significant differences between groups kept at high
on the development of preovulatory follicles only in the and low altitude (p < 0.05, Duncan’s test)

Table 2. Blood gases in sheep exposed to high altitude and the effect of antioxidant vitamins

Group PaO2 (mm Hg) PaCO2 (mm Hg) Ht (%) Hb (mg/dL) Sat Hb (%) pH

LL 97.5  0.8a 38.4  0.8a 29.1  1.5d 9.6  2.4c 96.1  2.1a 7.45  0.03
LLV 97.0  1.4a 37.7  1.6a 29.0  1.9d 10.2  0.6c 95.7  2.4a 7.47  0.02
LH 55.5  6.1d 29.1  3.6b 34.8  1.7a 13.8  0.9a 78.1  7.1d 7.49  0.03
LHV 57.1  3.4cd 25.9  2.7cd 32.7  1.4b 13.2  1.1a 80.4  3.0cd 7.47  0.11
HH 61.8  7.2b 26.3  2.8c 33.3  1.5c 12.1  1.1b 83.3  3.4b 7.49  0.03
HHV 59.6  6.1bc 24.4  1.9d 31.3  2.3b 13.1  0.8a 81.1  3.7bc 7.493  0.06

LL: low-altitude native sheep maintained at a low altitude, without vitamin supplementation; LLV: low-altitude native sheep maintained at a low altitude,
supplemented with vitamins; LH: low-altitude native sheep taken to a high altitude, without vitamin supplementation; LHV: low-altitude native sheep taken to a high
altitude, supplemented with vitamins; HH: high-altitude native sheep maintained at a high altitude, without vitamin supplementation; HHV: high-altitude native
sheep maintained at a high altitude, supplemented with vitamins.
Different superscript letters indicate significant differences among groups (p < 0.05).

© 2014 Blackwell Verlag GmbH

Preovulatory Follicle Development at High-Altitude 981

groups LL, LH and HH throughout the follicular phase.
However, the 17b-oestradiol concentrations reached
significantly higher values in the group LL than in
sheep exposed to high altitude (groups LH and HH) at
oestrous onset (p < 0.05).
The administration of antioxidant supplementation
showed no effects on the plasma 17b-oestradiol levels of
animals native to high altitude (group HHV) or native
of low altitude and maintained at low altitude (group
LLV) when compared with their non-treated counter-
parts (groups HH and LL). In any case, the values in
LLV sheep still remained higher than in groups HHV
and LHV.
Effects of altitudinal status and antioxidant treatment on
plasma FSH and LH concentration throughout the
oestrous cycle
The profile of daily plasma FSH concentration during
the entire oestrous cycle reproduces the wave-like
pattern driving follicular dynamics in all the groups,
without significant differences among them (Fig. 2). The
evaluation of the mean plasma FSH availability
1.6
HH
HHV
Plasma FSH (ng/ml)
throughout the oestrous cycle showed a significant
effect of the altitudinal origin, with significantly lower
values in the group HH than in the groups LH and LL
(p < 0.05, Table 3). The mean plasma FSH availability
during the ovulatory wave was numerically higher in the
group LH, although the differences were not statistically
significant (Table 4).
The antioxidant treatment showed no significant
effects on the mean plasma FSH concentration; differ-
ences among altitudinal origin were maintained
throughout the oestrous cycle, and the decrease in
FSH concentrations during the ovulatory wave found in
groups LLV and LHV was not statistically significant.
The profile of daily plasma LH concentration during
the entire oestrous cycle clearly shows an increase
around oestrous onset, during the periovulatory phase,
in all of the groups (Fig. 3). At this stage, the ewes
native to high altitude showed a significantly higher LH
concentration when compared to low-altitude native
ewes, either moved or not to high altitude (5.8  2.5 vs
1.3  0.8, p < 0.001). No significant effect of the
antioxidant administration was observed at this period
of the cycle. The same effect was observed in the mean
plasma LH availability throughout the oestrous cycle;
the group HH showed significantly higher values than
the groups LH and LL (p < 0.001, Table 5); at the same
time, the plasma LH concentration was lower in the

1.2
LH group LH than in the group LL (p < 0.05).
LHV The antioxidant treatment showed no significant
0.8 LL effects on the differences in the mean plasma LH
concentration among the groups HHV, LHV and
LLV LLV, as they were stable (p < 0.001). There were no
0.4 significant effects in any of the groups when compared
to their non-treated counterparts, in spite of numerical
0.0 increases in both sheep native and na€ıve to high altitude.
0 5 10 15
Day of the oeustrus cycle
Discussion
Fig. 2. Profile of daily FSH concentration throughout the entire
oestrous cycle. Day 0 = day of onset of the oestrus initiating the
The current experiment indicates that short-term expo-
studied cycle The different letters above columns indicate significant sure to hypobaric hypoxia in sheep newcomers to high
differences among groups (p < 0.05, Duncan’s test) altitude has a deleterious effect on both the ovarian

Table 3. Mean plasma FSH availability throughout the oestrous cycle (ng/ml)

HH HHV LH LHV LL LLV

0.65  0.05* 0.70  0.13 0.72  0.11 0.78  0.06 0.78  0.04 0.73  0.09

HH: high-altitude native sheep maintained at a high altitude, without vitamin supplementation; HHV: high-altitude native sheep maintained at a high altitude,
supplemented with vitamins; LH: low-altitude native sheep taken to a high altitude, without vitamin supplementation; LHV: low-altitude native sheep taken to a high
altitude, supplemented with vitamins; LL: low-altitude native sheep maintained at a low altitude, without vitamin supplementation; LLV: low-altitude native sheep
maintained at a low altitude, supplemented with vitamins.
Asterisk indicates significant differences with the other groups (p < 0.05).

Table 4. Mean plasma FSH availability during the ovulatory wave (ng/ml)

HH HHV LH LHV LL LLV

0.71  0.15 0.71  0.21 0.96  0.18 0.78  0.15 0.84  0.08 0.66  0.11

HH: high-altitude native sheep maintained at a high altitude, without vitamin supplementation; HHV: high-altitude native sheep maintained at a high altitude,
supplemented with vitamins; LH: low-altitude native sheep taken to a high altitude, without vitamin supplementation; LHV: low-altitude native sheep taken to a high
altitude, supplemented with vitamins; LL: low-altitude native sheep maintained at a low altitude, without vitamin supplementation; LLV: low-altitude native sheep
maintained at a low altitude, supplemented with vitamins.

© 2014 Blackwell Verlag GmbH

982 VH Parraguez, F Diaz, E Cofr
e, B Urquieta, M De Los Reyes, S Astiz and A Gonzalez-Bulnes

function (affecting preovulatory follicular development)
and the pituitary function (diminishing plasma LH
availability). On the other hand, there were no detected
differences in the preovulatory follicular development in
sheep adapted to high altitude for generations and,
conversely, LH secretion was increased, which suggests
an adaptive mechanism.
In the current experiment, all the animals in all the
groups ovulated; moreover, mean ovulation rates were
similar between groups. The sheep native from and
maintained at both low and high altitude (groups LL
and HH, respectively), showed profiles of follicular
growth which were similar to previously described
(Gonzalez-Bulnes et al. 2001, 2005). Most of the
preovulatory follicles arose from small antral follicles,
present at the moment of luteolysis, which grew during
the follicular phase for reaching ovulatory size and,
finally, ovulation. On the contrary, short-term exposure
to high altitude in na€ıve sheep (group LH) was related to
the presence of large follicles in static, or even early
atretic, growing phase at luteolysis which, despite
reaching ovulation, remained steady in size during the
follicular phase.
Adequacy or alterations in the development of
preovulatory follicles can be also assessed by determin-
ing their hormonal secretion. In sheep, oestradiol is
considered as a good marker of follicular quality
(Campbell et al. 1995; Gonzalez-Bulnes et al. 2004). In
the current study, plasma oestradiol levels were signif-
icantly lower in both sheep native and na€ıve to high
8
HH
6 bution of the hormones and growth factors to the
HHV
4 usually reach the ovulatory phase, whilst follicles with
Plasma LH (ng/ml)
altitude (groups HH and LH) than in ewes from group
LL, indicating alterations in the functionality of the
preovulatory follicles in sheep kept at high altitude.
These defective follicles, even ovulating and developed
in parallel with a functional corpus luteum, lead to
lower fertility (Ungerfeld and Rubianes 1999; Vi~ noles
et al. 2001), as their ovulation is related to alterations in
oocyte developmental competence, fertilization and
early embryo development (Revah and Butler 1996;
Mihm et al. 1999). Moreover, alterations in preovula-
tory follicle quality are known to be related to appear-
ance of defective corpora lutea previously described in
sheep exposed to high altitude (Parraguez et al. 2013).
Thus, the results of the current study strongly support
that alterations in follicle development would be a
definitive deleterious factor affecting fertility of sheep
na€ıve to high altitude. The fact that alterations in
follicular growth were found only after a short-term
exposure to hypobaric hypoxia would suggest an
immediate cause (i.e. alterations in the terminal devel-
opment of the preovulatory follicle rather than in the
quality of the ovarian follicle pool). This hypothesis is
reinforced by the fact that the supplementation with
antioxidant in a relative short period for the time of
folliculogenesis (approximately 1 month and a half)
changed substantially the development of preovulatory
follicles to similar patterns than in sheep native and
living to both high and low altitude. Oxidative stress
and, hence, antioxidants have been implicated in the
regulation of follicular development and ovulation (Al-
Gubory et al. 2010). With regard to the findings in the
current study, the ovulatory follicle is characterized by a
highly vascular theca layer (Redmer and Reynolds
1996), as a dense capillary network assures the distri-
follicular cells. In this way, highly vascularized follicles

LH
2 LHV
1 LL
LLV
0 ever, further studies are necessary for checking this
0 5 10
Day of the oestrous cycle
Fig. 3. Profile of daily plasma LH concentration throughout the
oestrous cycle. Day 0 = day of onset of the oestrus initiating the
studied cycle. Asterisk in the right panel indicates that group LH is
different from groups LHV, LL and LLV (p < 0.05, Duncan’s test)
reduced vascularization enter the atresia stage (Rey-
nolds et al. 2002). It is well known that hypoxia-induced
oxidative stress may activate the transcription of
vasoconstriction factors and may cause endothelial
dysfunction, but the administration of antioxidants
has been effective in the prevention of such vascular
dysfunctions (Herrera et al. 2014); therefore, a similar
effect may be hypothesized in the current study. How-
15 hypothesis.
At the same time, in the current study, sheep native to
low altitude but exposed to high altitude (group LH)
also showed lower plasma LH availability than sheep
native to and maintained at low altitude (group LL). In
sheep, like in other mammals, the gonadotrophin LH

Table 5. Mean plasma LH availability throughout the oestrous cycle (ng/ml)

HH HHV LH LHV LL LLV

0.62  0.60a 1.04  0.89a 0.13  0.04c 0.30  0.11b 0.28  0.18b 0.19  0.08b

HH: high-altitude native sheep maintained at a high altitude, without vitamin supplementation; HHV: high-altitude native sheep maintained at a high altitude,
supplemented with vitamins; LH: low-altitude native sheep taken to a high altitude, without vitamin supplementation; LHV: low-altitude native sheep taken to a high
altitude, supplemented with vitamins; LL: low-altitude native sheep maintained at a low altitude, without vitamin supplementation; LLV: low-altitude native sheep
maintained at a low altitude, supplemented with vitamins.
Different superscript letters indicate significant difference among groups (p < 0.05)

© 2014 Blackwell Verlag GmbH

Preovulatory Follicle Development at High-Altitude 983

appears to be the major luteotropin factor, playing a
critical role both for the development and maintenance
of the corpus luteum. In a previous study, the corpora
lutea of sheep na€ıve to high altitude were, overall,
smaller in size than the corpora lutea of ewes maintained
at low altitude (Parraguez et al. 2013). Thus, joining the
results of the previous and the present study, it can be
conclude that short-term exposure to high-altitude
hypobaric hypoxia affects pituitary LH secretion and,
in turns, corpus luteum development and, in conse-
quence, fertility.
The alterations in both LH secretion and follicular
dynamics found in the sheep na€ıve to high altitude in the
present study may be related. It is well known that the
largest growing follicles secrete inhibin and estradiol,
which depress FSH secretion, but these follicles avoid
their own regression in the absence of FSH by shifting
dependence from FSH to LH; hence, LH becomes
essential for the large dominant follicles (Campbell et al.
1995; Adams 1999). Thus, in case of low LH availability,
the development of these large follicles is compromised.
Moreover, the final maturation of the preovulatory
follicle is also dependent on LH, and hence, a low LH
availability may also interfere with this process. Further-
more, earlier studies on protocols for oestrous synchro-
nization evidenced that the alterations in patterns of LH
release gave way to lower quality of ovulations and lower
conception rates (Killian et al. 1985; Scaramuzzi et al.
1988). Failures in LH pulsatility may not be of enough
importance for avoiding ovulation and early luteogene-
sis, but may affect subsequent corpora lutea develop-
ment, as it was found in a previous study (Parraguez et al.
2013), and/or may lead to oocyte maturation weakness
and to limitations in the competence of these oocytes for
fertilization and subsequent embryo development (Ous-
said et al. 1999; Cogni
e et al. 2003).
On the other hand, sheep native to high altitude
(group HH) seem to have developed an adaptive
mechanism for counteracting the effects of exposure to
high-altitude hypobaric hypoxia; there were no apparent
deficiencies in the preovulatory follicular development,
and the plasma LH concentration was significantly
increased instead of diminished.
In conclusion, exposure of sheep to high-altitude
hypobaric hypoxia for short or long time periods
affects the final follicular growth and it steroidogenic
activity, which may be the cause of the previously
reported deficiencies in corpora lutea development and
subsequent fertility in these animals. In sheep na€ıve to
high altitude, preovulatory follicle development and
plasma LH availability were affected, which may
explain the decreased fertility observed after acute
exposure to high altitude. On the other hand, sheep
native to high altitude evidenced an adaptive mecha-
nism with adequate follicle dynamic and even
enhanced LH and slightly decreased FSH plasma
availability. Supplementation with antioxidant vita-
mins during a relative short period for the time of
folliculogenesis (approximately 1 month and a half)
changed substantially the development of preovulatory
follicles in high-altitude na€ıve sheep to similar patterns
than in sheep native and living to both high and low
altitude. These results highlight the role of oxidative
stress in the detriment of the reproductive function in
individuals recently exposed to high-altitude hypoxic
environment.
Acknowledgements
The authors thank Ms Laura P
erez for their technical assistance
during animal sampling and Mr Gabino Llusco for his assistance with
animal care. This work was supported by grant FONDECYT 1100189
from CONICYT, Chile and the Spanish Agency for International
Cooperation and Development (AECID; Technical Cooperation
Projects A/023494/09 and A/030536/10). AGB is member of the EU
COST-Action BM1308 ‘Sharing Advances on Large Animal Models
(SALAAM)’.
Conflict of interest
The authors confirm that this article content has no conflict of interest
that would prejudice the impartiality of the information.
Author contributions
All the authors contributed to the design of the study, the experimental
and assays procedures and the preparation of the manuscript.

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Submitted: 20 Jun 2014; Accepted: 6 Aug
2014
Author’s address (for correspondence): VH
Parraguez, Facultad de Ciencias Veterina-
rias, Universidad de Chile, Casilla 2, Correo
15, La Granja, Santiago, Chile. E-mail:
vparragu@uchile.cl
© 2014 Blackwell Verlag GmbH