Journal of Reproduction and Development, Vol. 51, No.

2, 2005

—Original—

Changes in Follicular Vascularity during the First Follicular

Wave in Lactating Cows
Tomas J. ACOSTA1)#, Ken-Go HAYASHI1), Motozumi MATSUI2) and
Akio MIYAMOTO1)
1)
Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro 080–8555, 2)Department of Clinical Veterinary Science, Obihiro
University of Agriculture and Veterinary Medicine, Obihiro 080–8555, Japan
#
Present: Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and
Technology, Okayama University, Okayama 700–8530, Japan

Abstract. Increase in the blood supply to individual follicles appears to be associated with follicular
growth rates and the ability to become the dominant follicle, while reduced thecal vascularity appears
to be closely associated with follicular atresia. Therefore, this study aimed to determine the real-time
changes in the vascularity of the follicle wall during the first follicular wave in cycling Holstein cows.
Normally cycling and lactating cows (n=5) were examined by transrectal color Doppler
ultrasonography (the sensitivity for velocity: > 2 mm/sec) to determine the changes in the vasculature
of the follicle wall (presence or absence of blood flow) and the diameter of follicles. A new follicular
wave and ovulation were induced by GnRH injection at 48 h after an injection of PGF2α analogue. The
ovaries were scanned daily for 7 days after GnRH injection. Follicles >2.5 mm were classified into 3
groups by the changes in diameter as follows: 1) largest follicle, 2) second largest follicle, and 3) small
follicles, which included all other follicles >2.5 mm. Before the follicle selection, there was no
significant difference in the percentage of follicles with detectable blood flow between the
subsequently determined largest and second largest follicles. After the follicle selection, the
percentage of follicles with detectable blood flow significantly decreased among the second largest
follicles. In addition, small follicles with detectable blood flow kept larger diameters than those
without detectable blood flow from one day before the occurrence of follicle selection. It is likely that
maintenance of follicle vasculature and appropriate blood supply to the larger follicles is essential for
follicle dominance. In small follicles, the presence of blood flow within the wall also appears to be
required for recruitment. Consequently, the data suggest that the change of the blood supply to an
individual follicle closely relates to the dynamics of follicular growth in the first follicular wave in the
cow.
Key words: Blood flow, Follicular wave, Selection, Deviation, Cow
(J. Reprod. Dev. 51: 273–280, 2005)

election of the dominant follicle in cattle occurs first wave (wave 1) appears around the time of
from a cohort of growing antral follicles, termed ovulation and the second during mid-diestrus
a follicular wave [1–3]. Two or three successive (wave 2). Following emergence of a group of
waves occur during the estrous cycle in cattle. The follicles 4 to 5 mm diameter, the follicles develop in
a common-growth phase for about 3 days. When
Accepted for publication: December 28, 2004
Published online: February 3, 2005 the largest follicle reaches a mean diameter of about
Correspondence: A. Miyamoto (e-mail: [email protected]) 8.5 mm, the common-growth phase ends and
274 ACOSTA et al.

deviation in diameter begins, characterized by the daily changes in blood flow within the follicle
dissociation in growth rates and the formation of wall around the time of diameter deviation and
dominant and subordinate follicles [3]. After the demonstrated considerable differences in blood
end of the common growth phase, only the selected flow area between the two largest follicles one day
dominant follicle continues growing, while before diameter deviation [13]. The mean diameter
subordinate follicles undergo atresia [4]. The of the largest follicle at deviation is about 22.5 mm
dominant follicle in the cow is normally identified in mares and about 8.5 mm in cattle [3]. The
when it reaches a diameter of 10 mm and is larger smaller follicular size at deviation in cattle
than the other follicles of the same wave. At this represents a technical limitation for determination
size, the dominant follicle acquires LH receptors on of blood flow velocities, although blood flow
its granulosa cells and acquires ovulatory capacity velocity has been accurately determined in healthy
in response to the LH surge [5]. growing follicles of more than 10 mm of diameter
During follicle growth, an extensive vascular [11].
plexus develops in the theca layer surrounding the The physiological mechanisms involved in the
avascular basement membrane and granulosa layer selection of the dominant follicle from identical
[6]. Morphological studies of bovine follicles have follicular cohorts exposed to the same levels of
revealed that the major histological characteristic of circulating gonadotropins remain unclear. It has
the selected (dominant) follicle is its increased been suggested that the preferential delivery of
vascularity in the theca layer, when compared with gonadotropins and nutrients via a more highly
unselected (atretic) follicles [7]. It has been developed vascular system in individual follicles
suggested that insulin-like growth factor and plays a role in the selection and growth of the
estradiol (E2) play key roles in the selection of a dominant follicle [14–17]. The present study was
dominant follicle during the first wave in heifers carried out to determine if color Doppler
and that both are reliable markers to predict the ultrasonography could distinguish future
future dominant follicle [8]. Other studies have dominant follicles from subordinate follicles
demonstrated a close correlation between around the time of the establishment of follicle
intrafollicular concentration of E 2 and follicular selection (deviation), and whether or not there is a
vascularization in porcine and bovine ovaries [7, 9]. temporal association between changes in follicular
However, studies on the differences among follicles vascularity and follicular growth capacity during
in terms of vascularity and growth rates around the the first follicular wave in lactating cows.
time of follicle selection (deviation) have not been
carried out in cattle.
Color Doppler ultrasonography is a useful, non- Materials and Methods
invasive tool for evaluating the vascular function of
follicles, allowing visual observation of the blood Animals and ultrasound scanning
flow in a delimited area in the wall of preovulatory The animal experiment was carried out at the
follicles in humans [10] and cows [11]. In human, Field Center of Animal Science and Agriculture,
blood flow determinations of individual follicles by Obihiro University, Japan. Experimental
Doppler ultrasound provide an index of the procedures complied with the Guide for Care and
intrafollicular environment and may be used to Use of Agriculture Animals of Obihiro University.
predict the developmental competence of the Lactating Holstein cows were kept under the
oocyte [12]. In cows, transrectal color Doppler normal management program of the Field Center
ultrasonography demonstrated a clear difference in and fed daily with corn silage, hay and concentrate
the vascularity of the wall of preovulatory follicles with free access to water. At the middle stage of the
compared with anovulatory follicles [11]. The estrous cycle, cows received 500 µg of a
results of the above reports suggest that transrectal prostaglandin (PG) F 2α analogue (cloprostenol
Doppler ultrasonography has potential for [estrumate]; Sumitomo Pharm. Co., Osaka, Japan)
investigating follicle vasculature in cattle to i.m. to induce luteolysis. A new follicular wave and
identify the future dominant follicle at an early ovulation of the preovulatory follicle was induced
development stage or to predict follicle viability by a GnRH analogue (Fertirelin acetate 100 µ g;
after selection. A recent study in mares quantified [Conceral]; Nagase Pharm. Co., Osaka, Japan)
 LOCAL BLOOD FLOW DURING FOLLICULAR DEVELOPMENT 275

injected 48 h after PGF 2 α . The day of GnRH Progesterone and estradiol determinations
injection was designated as Day 0. Blood samples were obtained by caudal
Ultrasound scanning and blood collection were venipuncture just before each scanning using
performed daily beginning on the day of PGF 2α sterile 10 ml tubes containing 200 µl of stabilizer
treatment and continued until Day 7. To determine solution (0.3 M EDTA, 1% acetyl salicylic acid, pH
changes in the follicular diameter and follicle 7.4). All tubes were immediately chilled in ice
vascularity, color Doppler ultrasonography was water for 10 min. and then, centrifuged at 3000 × g
used. The follicles were examined by transrectal for 20 min at 4 C. Aspirated FF was centrifuged at
ultrasonography using an ultrasound scanner 1000 × g for 5 min to remove granulosa cells. The
(SSD-5500, Aloka Co., Tokyo, Japan) equipped with obtained plasma and FF were stored at –30 C until
a 7.5-MHz convex transducer (UST-995–7.5, Aloka determination of the concentrations of P4 and E2.
Co.). All follicles >2.5 mm present in both ovaries At the end of the experiment, samples from each
were tracked daily during the examination period. cow were analyzed in duplicate using a second
The two largest follicles were defined antibody enzyme immunoassay (EIA). Steroids
retrospectively according to the maximum attained assays w er e p er fo rm ed afte r d i ethyl et he r
diameter and were then used to define the day of extraction. For E2 determination, FF was diluted
deviation. During each ultrasonographic 50- or 500-fold so that concentrations in the EIA
examination, the diameter of each follicle was would be within the optimal range of the standard
determined by averaging the maximum and the curve as reported previously for our laboratory
transverse diameters as previously reported [13]. [18]. The standard curve for P4 ranged from 0.05 to
After morphological evaluation, the flow mode was 50 ng/ml, and the ED50 of the assay was 1.1 ng/ml
activated for blood flow mapping. Color signals [19]. The intra- and inter-assay coefficients of
were used to generate images in which blood flow variation (CVs) were 4.7 and 6.5%, respectively.
with a velocity higher than 2 mm/sec could be The recovery rate of P4 (1 ng) added to 1 ml plasma
located as areas of color within the follicle wall. samples was 92% (n=10). Likewise 40 µl of samples
Forward flow is usually presented in red and were analyzed for E2. The standard curve ranged
reverse flow in blue. The degree of turbulence is from 2 to 2000 pg/ml, and the ED50 of the assay was
indicated as color-coded signal. The brightness of 72 pg/ml. The intra- and interassay CVs were 6.8
the color is proportional to the velocity of flow and 8.6%, respectively.
within the vessel. Owing to technical limitations
for measurement of blood velocities in small Statistical analysis
individual follicles, only the presence or absence of The diameters of follicles were expressed as
blood flow was assessed for each follicle. When a means ± SEM, and compared with values at the
clearly visible red or blue spot (blood flow) was same time point by repeated measures ANOVA
detected in the follicle wall, it was considered as a followed by Student’s t test. The percentages of the
follicle with detectable blood flow. follicles with detectable blood flow in largest,
Scan records (images) were stored on a Magneto second largest and small follicles were compared
Optical (MO) disk drive for a personal computer before and after follicle deviation using two-way
(Macintosh; Apple Corp., San Jose, CA) and then ANOVA followed by Fisher’s PLSD test.
viewed on a monitor. The same image was used to Differences were considered significant at p<0.05.
calculate the diameter and whether or not the blood
flow was detectable. After the last ultrasound
scanning on Day 7, the dominant follicle content Results
was evacuated by transvaginal ultrasound-guided
follicular aspiration using a convex probe (UST- The GnRH injection induced ovulation of the
M 15 - 21 0 79 A l o k a C o . ) w i t h an 1 8G n e e d l e preovulatory follicle and the emergence of a new
connected to a 5-ml disposable syringe. Follicular follicular wave in all the cows used in the present
fluid (FF) was collected to determine the study. Plasma P 4 concentrations progressively
concentrations of progesterone (P4) and E2. increased throughout the experiment with CL
development (Fig. 1). The mean number of follicles
tracked during the examination was 1 dominant
276 ACOSTA et al.

Fig. 2. Changes in the diameter of the largest

Fig. 1. Daily changes in the plasma follicle ( ) and second largest follicle ( ).
concentration of progesterone after Data were normalized to the day of
GnRH injection. Data are mean ± SEM observed deviation (Day 0). Data are
concentrations of circulating mean ± SEM for each time point (n=3–5). *
progesterone (n=5 cows). p<0.05, ** p<0.01 and *** p<0. 001 versus
values of second largest follicles.

follicle and 7 subordinate follicles per cow. A clear healthy and dominant on Day 7.
deviation in the growth rates between the two
largest follicles occurred between 3 and 4 days after Variation of the blood flow within the follicle wall
GnRH injection. Because the length of time from Time-average maximum velocity could be
GnRH injection to follicle deviation varied among measured in some large follicles on each day, but
the cows, the data were normalized to the day of the follicles in which velocity could be measured
deviation based on the criteria of a previous study varied from day to day with values ranging from
[5]. Briefly, the beginning of diameter deviation 4.1 cm/sec to 10.4 cm/sec during the growth phase.
was defined as the beginning of the greatest Therefore, only the presence of blood flow was
difference in growth rates between the largest used to evaluate follicular vascularity in the present
follicle and second largest follicle (observed study. The color Doppler image detected blood
deviation). Follicles were classified retrospectively flow in the follicle wall of most of the largest and
into 3 groups based on the changes in diameter as second largest follicles and some small follicles
follows: 1) the largest follicle, which grew during the growth phase (Fig. 3). After deviation
continuously and attained the maximum diameter had occurred, the blood flow was mainly detectable
during the examination period; 2) the second in the largest follicle.
largest follicle, which grew for 3 to 4 days up to the
day of deviation; and 3) small follicles, which other Changes in the percentages of follicles with detectable
follicles. The mean diameters of the two largest blood flow during follicle deviation
follicles from Day –3 to Day 3 normalized to the Percentages of follicles with detectable blood
day of observed deviation are shown in Fig. 2. The flow during follicle deviation are shown in Fig. 4.
largest follicles continuously grew up to 14.1 ± 0.6 Before the occurrence of diameter deviation (pre-
mm on Day 3. The diameter of the second largest deviation), there was no significant difference in
follicle increased from 5.1 ± 1.1 mm on Day –3 to 8.3 the percentages with detectable blood flow
± 0.6 mm on Day 0 and then decreased to 7.7 ± 0.9 between the two largest follicles. In small follicles,
mm on Day 3. Concentrations of E2 and P4 in FF the percentage of follicles with detectable blood
aspirated from dominant follicles were 63.0 ± 21.4 flow was lower than that for the future largest
ng/ml and 26.0 ± 6.9 ng/ml, respectively. follicle and future second largest follicle. After the
Accordingly, the ratio of E2 to P4 concentration in follicle deviation (post-deviation), the percentage of
FF was 2.4 ± 0.5 (n=5) confirming that the largest follicles with detectable blood flow significantly
follicles classified by change in diameter were decreased in second largest follicle compared with
 LOCAL BLOOD FLOW DURING FOLLICULAR DEVELOPMENT 277

Fig. 3. Representative color Doppler images of the ovary around the day of follicle deviation.
Encircled images showing largest (upper panel), second largest (middle panel) and small
follicle (lower panel) at Days –2, 0 and 3 from follicle deviation. Enlarged follicle images
of the encircled part showing typical changes in detectable blood flow. Arrow indicates
areas with detectable blood flow within the follicle wall. Scale bar represents 1 cm.

values before deviation. During the post-deviation

period, the percentage of follicles with detectable
blood flow in the largest follicles was significantly
higher than in the second largest follicles and small
follicles.

Changes in the diameter of small follicles with

detectable blood flow and undetectable blood flow
To clarify further the effect of blood flow on the
development of small follicles, small follicles were
further classified as small follicles (+), which
showed detectable blood flow at least once during
the examination period, and small follicle (–),
which did not show any blood flow during the
examination period. The mean diameter of small Fig. 4. Change in the percentage of follicles with
follicle (+) was significantly larger than that of detectable or undetectable blood flow around the
day of follicle deviation. Data are mean ± SEM of
small follicle (–) from Day -1 to Day 3 (Fig. 5). each group of follicles. Different letter indicate
differences (p<0.05) between groups.
278 ACOSTA et al.

nutrients and gonadotropins provided by a

developing vascular system equally contribute to a
similar growth rate in both future dominant and
subordinate follicles before follicle deviation.
After follicle deviation, our data show that the
percentage of largest follicles with detectable blood
flow was significantly higher than that of the
second largest follicles and small follicles. It has
been demonstrated that the capillary blood flow
velocity of the dominant follicle is greater than that
of the atretic follicles in ewes [21]. These results
strongly suggest that blood supply is preferentially
Fig. 5. Changes in the follicle diameter of small delivered to the dominant follicle rather than
follicles with detectable blood flow ( ) subordinate follicles after diameter deviation. It is
and with undetectable blood flow ( )
evident that the dominant follicle maintains blood
around the day of follicle diameter
deviation. Data are mean ± SEM for each vessel and active angiogenesis [7, 22]. In bovine
time period (n=6–20 follicles). *** p<0.001 follicles, the expression of mRNA for angiogenic
versus values of small follicles with factors such as vascular endothelial growth factor
detectable blood flow.
(VEGF) and basic fibroblast growth factor, and
concentration of VEGF protein in FF significantly
increased accompanied with increasing follicular
Discussion size and E2 levels in FF [14]. In addition, a recent
report suggested that the angiopoietin-Tie system
To the best of our knowledge, this is the first that acts on angiogenesis in concert with VEGF
report on real-time changes in follicle vascularity controls the blood vessels to maintain active
associated with the selection of the dominant angiogenesis in developing bovine follicles [23]. In
follicle during the first follicular wave in lactating contrast, degenerative thecal capillary blood
cows. We used highly sensitive (2 mm/sec in vessels are more abundant and the number of
velocity) color Doppler ultrasonography to identify blood vessels is markedly reduced in atretic
blood flow area in the follicle wall. The ultrasound- follicles [6, 7]. These findings indicate that active
derived images of blood flow revealed the time- angiogenesis ensuring blood vessel network and
related changes in appearance of detectable local blood supply to the follicle may play a crucial role
blood flow in the largest, the second largest and in the determination of the follicle dominance and
small follicles during the first wave. atresia.
In the present study, the percentage of follicles Quite recently, the daily observation of equine
with detectable blood flow before diameter ovaries using color Doppler ultrasonography
deviation was not different between the largest and demonstrated that velocities and the area of blood
second largest follicles. Morphological study has flow within the follicle wall began to decrease in
shown that bovine healthy antral follicles with the future subordinate follicle prior to deviation in
diameters of 3 to 7 mm develop dense capillary diameter, whereas the blood flow continued to
blood vessel networks in the theca layer [7]. These increase in the future dominant follicle during
results suggest that there is no difference in follicle selection [13]. In addition, mares with
vasculature and blood supply between the future double dominant follicles showed no differences in
dominant and subordinate follicles before follicle diameter and blood flow in the follicle wall
deviation. Furthermore, there is also no difference between the two largest follicles [13]. These data
between each follicle in levels of mRNA for FSH from mares and the present results from cows
receptor in the granulosa cells or mRNA for LH support the concept that different blood flow
receptor in theca cells up to follicle deviation [20], changes within the follicle wall influence the fate of
suggesting that there is no difference in the follicles and that detectable blood flow and
responsiveness to gonadotropins between the two vasculature are associated with follicle viability.
largest follicles at this stage. Equivalent supply of It is interesting to note that the diameter of small
 LOCAL BLOOD FLOW DURING FOLLICULAR DEVELOPMENT 279

follicles with detectable blood flow was larger than is likely that maintenance of follicle vasculature
those without detectable blood flow before follicle and appropriate blood supply to the follicle is
deviation. The present result agrees with a recent essential for follicle dominance. In small follicles,
observation of non-dominant follicles during the the presence of blood flow within the wall appears
luteal phase in which healthy follicles with 6.7 mm to be required for recruitment. Consequently, the
of mean diameter showed active angiogenesis, and data suggest that a change of the blood supply to
that atretic small follicles with a mean diameter of individual follicles closely relates to the dynamics
4.8 mm showed poorly developed capillaries in the of follicular growth in the first follicular wave in the
follicle wall [7]. These results indicate that small, cow.
but healthy growing follicles maintain angiogenesis
and blood flow during early follicular
development. Recent studies have demonstrated Acknowledgments
that modification of follicular angiogenesis by
inhibition of VEGF action or stimulation of VEGF The authors thank Dr. K. Okuda (Okayama
production caused a delay or induction of follicular University, Japan) for progesterone antiserum.
development, respectively [17, 24]. Taken together, This study was supported by a Grant-in-Aid for
we propose that angiogenesis and blood supply in Scientific Research of the Japan Society for the
follicles may be involved in not only selection of the Promotion of Science (JSPS), and the 21st Century
dominant follicle but also early follicular COE Program (A-1) of the Ministry of Education,
development including follicular recruitment. Culture, Sports, Science, and Technology, Japan.
In conclusion, our results demonstrate that T.J.A. and M.M were postdoctoral fellows
change in the number of follicles with detectable supported by JSPS and the COE Program,
blood flow was associated with follicle selection. It respectively.

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