Journal of Reproduction and Development, Vol. 53, No. 6, 2007, 19057—Full Paper—

Follicle Size-Dependent Changes in Follicular Fluid
Components and Oocyte Diameter in Antarctic Minke Whales(Balaenoptera bonaerensis)
Hiroki NAGAI1), Toshihiro MOGOE2), Hajime ISHIKAWA2), Shinichi HOCHI3),
Seiji OHSUMI2) and Yutaka FUKUI1)
1)

Laboratory of Animal Reproduction, Obihiro University of Agriculture and Veterinary
Medicine, Obihiro 080-8555, 2)The Institute of Cetacean Research, Tokyo 104-0055 and3)Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567, Japan
Abstract. The concentrations of various components of follicular fluid were compared among threegroups of follicles (small, <5 mm; medium: 5–10 mm; large, >10 mm) with a control that consisted of
the components of umbilical serum using seven pregnant Antarctic minke whales. Follicular oocytes
recovered from the follicles were also used for measurement of oocyte diameter after removing the
cumulus cells. The mean diameter of the ooplasm in the oocytes from the large follicles (143.2 µm)
was significantly greater than those from the small (127.1 µm) and medium (131.7 µm) follicles,
although there were no significant differences in diameter of the whole oocyte and thickness of thezona pellucida among the three follicular sizes. The osmolarity of the follicular fluid from the smallfollicles (363.3 mOsmol) was significantly lower than that of the medium follicles (388.9 mOsmol) andtended to be lower than that of large (381.9 mOsmol) follicles, respectively, both of which were similar
to that of the umbilical serum (379.5 mOsmol). There was no significant difference in theconcentrations of all components of the follicular fluid between the medium and large follicles. As
compared with the values of the umbilical serum, the total-protein, glucose, albumin and chlorine
concentrations of the follicular fluid from the medium and large follicles were significantly higher,
and the total cholesterol and calcium concentrations were significantly lower. The concentrations oflactic acid (85.3–136.0 mg/dl) of the follicular fluid from the three groups of follicles were significantly
lower than that of the umbilical serum (360.0 mg/dl). Among the follicles, the follicular fluid from thesmall follicles (136.0 mg/dl) contained a significantly higher concentration of lactic acid than that fromthe large follicles (85.3 mg/dl). The progesterone concentrations were not significantly different
among the fluid from the three group of follicles and the umbilical serum: however, the estradiol 17-β
concentrations of the follicular fluid increased with the size of the follicle (14.3 and 34.6 ng/ml forsmall and large follicles, respectively). These results offer new information concerning whale
reproductive physiology, especially for improvement of in vitro oocyte maturation and related
technologies in whales.
Key words: Follicular fluid, Follicle size, Minke whale (Balaenoptera bonaerensis), Oocyte diameter(J. Reprod. Dev. 53: 1265–1272, 2007)

tudies of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of
Accepted for publication: August 29, 2007
Published online: October 23, 2007
Correspondence: Y. Fukui (e-mail: fukui@obihiro.ac.jp)

Antarctic minke whale (Balaenopetera bonaerensis)
oocytes have been performed in our laboratory [1–3]. Iwayama et al. [4] reported a successful shortening of the IVM culture period for fresh minke whale
oocytes from 120 to 30 h by adding pregnant whale

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follicular fluid (wFF) to an IVM medium in place of
supplementation with calf or whale serum. Furthermore, by adjusting the osmolarity of the IVM
medium to 390 mOsmol, Fujihira et al. [5] improved
the IVM rates for freshly collected immature
oocytes from adult and prepubertal Antarctic
minke whales (60.9 and 53.1%, respectively). These
results indicate the importance of adding follicular
fluid (FF) collected from pregnant females to IVM
medium for whale immature oocytes. It has
recently been suggested that chemically defined
culture media can be used for IVM and IVC inorder to clarify the effectiveness of various additives in the media, such as hormones and growth
factors [6–9]. However, FF has been widely demonstrated to have a positive effect on IVM culture ofvarious mammalian oocytes, and it has been shownthat addition of FF during IVM enhances the malepronuclei formation of oocytes after IVF and subsequent embryonic development [10–14].
Furthermore, FF collected from larger follicles contains some proteins and higher expression ofmRNA that improve the progression of nuclear andcytoplasmic maturation and the developmental
competence of bovine and porcine oocytes after IVF[13, 14]. The relationships between follicular sizeand oocyte developmental capacity have been
established for many species, including cattle [15],goats [16], pigs [17] and buffaloes [18]. It has also
been reported the developmental competence isaffected by oocyte diameter and that the oocytes
recovered from smaller follicles are generally
smaller in diameter and less competent comparedwith those recovered from larger follicles [19–22].
In regard to whales, Fukui et al. [23] and Suzuki etal. [24] have previously measured the plasma or
serum concentrations of gonadotropins and steroid
hormones in pregnant and non-pregnant Antarctic
minke whales. However, the concentrations of thechemical components of wFF from follicles of different sizes have not been investigated in detail,except for some steroid hormones such as progesterone (P4), testesterone (T) and estradiol 17-β (E2)[25]. In our previous studies [4, 5], wFFs collectedfrom various sizes of follicles from pregnant
females were pooled and used for IVM culture of
Antarctic minke whale oocytes. If only wFF collected from larger follicles in IVM culture positively
affect IVM and subsequent embryonic development, as shown in several studies of domestic
animals [13, 14], analyses of wFF components could

contribute the design of a chemically defined IVM
medium for whale oocytes.
Therefore, the present study was conducted tocompare the concentrations of various components
of wFF collected from follicles of different sizes(small, <5 mm; medium, 5–10 mm; large, >10 mm)
with a control that consisted of the components ofumbilical serum using seven pregnant Antarctic
minke whales. Vitrified and warmed follicularoocytes derived from the three groups of follicles
were also used for measurement of the diameters of
the whole oocyte, ooplasm and zona pelllucida
after removing the cumulus cells.

Materials and Methods
WhalesThe Antarctic minke whales used in this study
were obtained from November 2005 to March 2006
as part of the Japanese Whale Research Programwith a Special Permit for the Antarctic (JARPA),
which was organized by the Institute of Cetacean
Research (Tokyo, Japan). The seven pregnant
minke whales (body length: 9.04 ± 0.10 m) usedwere obtained in an area 60 degrees south to the iceedge and between 130 degrees east and to 145
degrees west. All the minke whales possessed apregnant corpus luteum in one of their ovaries. The
fetal length ranged from 60.4 to 149.5 cm (mean ±SEM: 118.1 ± 12.3 cm). The whales were killed with
explosive harpoons, which have been recognizedby the International Whaling Commission as themost humane method for killing whales, as provided for by Schedule III (C apture) of theInternational Convention for the Regulation of
Whaling. Special efforts were made to reduce thetime to death for all sampled whales. Explosive
harpoons were used for all whales as the primary
killing method. A large-caliber rifle was used as
the secondary killing method as required. The ovaries were collected within 3 h after death, and were
kept warm at 20–25 C until oocyte collection(within 8 h).

Oocyte collection and vitrification
Cumulus-oocyte complexes (COCs) were aspirated from three groups of follicles (<5 mm, small; 5
to 10 mm, medium; >10 mm, large in diameter) of
the minke whales using a 10-ml syringe fitted withan 18-gauge needle. The oocytes surrounded by

FOLLICLE SIZES OF ANTARCTIC MINKE WHALE

more than two layers of cumulus cells and withhomogeneous cytoplasm were separately selected
and washed three times in Medium 199 (M199,
Sigma-Aldrich Chemical, St. Louis, MO, USA) containing 0.1% (w/v) polyvinyl alcohol (PVA, Sigma),
2 mM NaHCO 3 , (Wako Pure Chemicals, Osaka,
Japan), 10 mM HEPES (Sigma) and 75 mg/l kanamycin (Sigma; M199-PVA).
The oocytes were essentially vitrified-warmed
according to the method of Fujihira et al. [5]. In
brief, the COCs were transferred to M199-20% (v/v) newborn calf serum (NBCS; Gibco-BRL, Grand
Island, NY, USA; M199-NBCS, osmolarity, 270
mOsmol) containing 7.5 µ g/ml cytochalasin B(Sigma) for 30 min. The COCs were then exposed
to 7.5% cryoprotectant (3.75% ethylene glycol, EG,
and 3.75% dimethyl sulfoxide, DMSO) in M199NBCS for 8 min and then 15% cryoprotectant (7.5%
EG and 7.5% DMSO) in M199-NBCS for 4 min at 37
C. Finally, the COCs were exposed to 30% cryoprotectant (15% EG and 15% DMSO) and 0.5 M sucrose(Wako) in M199-NBCS. Within 1 min, 3 to 5 COCs
were placed on a sheet of cryotop (Kitazato Supply,Tokyo, Japan) [5, 26], which was then immersed inliquid nitrogen. For warming, the cryotop sheet
holding the COCs was immersed in M199-NBCS
containing 0.5 M sucrose at 37 C for 5 min and wasfurther diluted by exposure to M199-NBCS containing 0.25 and 0.125 M sucrose for 2 and 2 min,respectively, at 37 C.
After mechanical removal of cumulus cells fromthe post-warm COCs, the diameter of the whole
oocyte including the zona pellucida, diameter ofthe ooplasm and thickness of the zona pellucida
were recorded by means of two perpendicular measurements. Ten oocytes derived from each groupof follicles (small, medium and large) were used forthe measurement.

Follicular fluid and umbilical serum
The FF was aspirated from the three groups offollicles (small, medium and large) for collection ofoocytes from the seven pregnant whales and was
individually stored in 1.8 ml cryo-tubes at –20 C.
Umbilical blood was collected from the fetal cords
of four out of the seven pregnant whales, and thesamples were stored for 1 h at 4 C to allow clotting.
The samples were centrifuged (600 g × 20 min) toseparate the serum and were kept at –20 C until
analyses.

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Measurement of the concentrations of follicular fluid
and umbilical serum components
Osmolarity: The osmolarity of the FF from thethree groups of follicles (n=14 for small, n=22 for
medium and n=24 for large) and the umbilicalserum (n=4) was measured using an osmometer(OM801, Vogel, Giessen, Germany). Each sample
was measured two times.
Chemical components: The concentrations of
total-protein (TP, g/dl), total-cholesterol (T-CHO,
mg/dl), glucose (GL, mg/dl), albumin (ALB, g/dl),
sodium (Na, mg/dl), potassium (K, mg/dl), chlorine (Cl, mg/dl), calcium (Ca, mg/dl), phosphate(P, mg/dl) and magnesium (Mg, mg/dl) in the FFof the medium (n=20) and large (n=20) follicles andthe umbilical serum (n=4) were measured at theTokachi Clinic Center (Obihiro, Japan); TP wasmeasured by a Biuret method, ALB was measured
by a bromocresol green method, T-CHO and GL
were measured by an enzyme method, Na, K and
Cl were measured by an electric-rod method, Ca
was measured by an o-cresolphthalein complexone
method, Mg was measured by a xylidyl blue
method and P was measured by an molybdenumdirect method.
Due to the limited sample volume, the chemicalcomponents in the FF collected from the small follicles could not be obtained for measurement withexception of lactic acid. The concentration of lactic
acid was measured by an enzyme assay using acommercial kit (Determiner LA; Kyowa-Hakkou,
Osaka, Japan). The intra- and interassay coefficients of variation were 3.0 and 1.9%, respectively.
Each sample was measured two times.
Steroid hormones: The concentrations of P4 andE2 in the FF from the three groups of follicles (n=18
for small, n=25 for medium and n=25 for large) andthe umbilical serum (n=4) were measured byenzyme immunoassay as reported in our previous
studies [24, 25]. The intra- and interassay coefficients of variation were 5.2 and 9.3% and 4.8 and8.6% for P4 and E2, respectively. Each sample wasmeasured two times.

Statistical analysis

All data are presented as means ± SEM. KruskalWallis tests and the Scheffe method using the StatView program (Abacus Concepts, Berkeley, CA,
USA) were used to determine differences for eachcomponent of the wFF collected from the threegroups of follicles (small, <5 mm; medium, 5–10

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NAGAI et al.

Table 1. Diameters (µm, mean ± SEM) of the whole oocyte and ooplasm of the Antarctic minke whales
Follicular size (mm)
5–10

<5
n
Whole oocyte
Ooplasm
Zona pellucida

1

>10

1

10*1
188.7 ± 2.5
143.2 ± 3.1b22.7 ± 0.8

10*184.4 ± 3.0
131.7 ± 3.7a26.3 ± 1.4

10*179.2 ± 3.1
127.1 ± 3.1a
26.1 ± 1.6*1 Numbers of follicles examined. a, b The different superscripts indicate significant differences (P<0.05).

Table 2. Osmolarity (mOsmol, mean ± SEM) of the follicular fluid derived from the different sizes of thefollicles of the Antarctic minke whalesnOsmolarity<5

Follicular size (mm)
5–10

<10

Umbilical serum

14*1
363.3 ± 4.2a

22*1
388.9 ± 6.6b

24*1
381.9 ± 4.4a,b

4*2
379.5 ± 5.8a,b*1 Numbers of follicles examined. *2 Numbers of fetuses examined. a, b The different superscripts indicate significant differences (P<0.05).

mm; large, >10 mm) and the umbilical serum.
Groups producing P values <0.05 were consideredsignificantly different.

Results
Diameters of the whole oocyte and ooplasm
As shown in Table 1, the diameters of the ooplasm of the oocytes recovered from the small (127.1± 3.1 µ m) and medium (131.7 ± 3.7 µ m) follicles
were significantly smaller (P<0.05) than that of theooplasm of the oocytes recovered from the large
follicles (143.2 ± 3.1 µm), although there was no significant difference in diameter of the whole oocyte
and thickness of zona pellucida among the threefollicular size groups.

Concentrations of chemical components in follicular
fluid and umbilical serum
The concentrations of various components of the
FF collected from the medium and large follicles
and umbilical serum are shown in Table 3. There
were no significant differences in the concentrations of any of the components of the wFF collectedfrom the medium and large follicles. However, theTP, GL, ALB and Cl concentrations of the FF fromthe medium and large follicles were significantly
higher (P<0.05) and T-CHO and Ca concentrations
were significantly lower (P<0.05) than those of theumbilical serum. The concentrations of the othercomponents of the FF collected from these twogroups of follicles and the umbilical serum were
not significantly different.

Lactic acid and steroid hormone concentrations
Osmolarity of follicular fluid and umbilical serum
As shown in Table 2, the osmolarity of the FF of
the small follicles (363.3 ± 4.2 mOsmol) was significantly lower than that of the FF collected from themedium follicles (388.9 ± 6.6 mOsmol; P<0.05) andtended to be low compared with the large follicles(381.9 ± 4.4 mOsmol), which had a value similar tothat seen in the umbilical serum (379.5 ± 5.8
mOsmol).

As shown in Table 4, the concentrations of lactic
acid in the FF collected from the three groups of follicles were significantly (P<0.05) lower than that in
the umbilical serum. Among the follicles, the FF
collected from the small follicles (136.0 ± 16.1 mg/dl) had a significantly higher concentration of lactic
acid than that collected from the large follicles (85.3± 10.0 mg/dl: P<0.05).
The P4 concentrations were not significantly different among the three groups of follicles and theumbilical serum, while the E2 concentrations of thewFF increased as the size of the follicles increased

FOLLICLE SIZES OF ANTARCTIC MINKE WHALE

1269

Table 3. Concentrations of various components of follicular fluid and umbilical serum (mean ± SEM) of
the Antarctic minke whales
Components

Follicular size (mm)nTotal-protein (g/dl)
Total-cholesterol (mg/dl)
Glucose (mg/dl)
Albumin (g/dl)
Na (mg/dl)
K (mg/dl)
Cl (mg/dl)
Ca (mg/dl)
P (mg/dl)
Mg (mg/dl)

Umbilical Serum

5–10

>10

20*1
5.2 ± 0.1a
101.2 ± 6.0a
82.6 ± 4.8a
3.8 ± 0.1a
387.8 ± 7.4
48.9 ± 2.7
439.7 ± 15.7a
8.5 ± 0.3a
9.6 ± 0.2
3.9 ± 0.3

20*1
5.3 ± 0.1a
102.7 ± 6.7a87.9 ± 5.7a
3.8 ± 0.1a
387.0 ± 6.8
45.1 ± 2.9
435.9 ± 13.0a
9.7 ± 0.5a
10.6 ± 0.5
3.9 ± 0.3

4*2
3.7 ± 0.2b
159.0 ± 4.1b
3.8 ± 0.9b
2.7 ± 0.1b354.2 ± 2.8
54.1 ± 3.3
330.2 ± 3.8b
15.1 ± 0.4b11.3 ± 0.4
4.5 ± 0.2

*1, *2 See the footnotes of Table 2. a, b The different superscripts indicate significant differences (P<0.05).

Table 4. Concentrations of lactic acid and steroid hormones in the umbilical serum and follicular fluid fromdifferent sizes of follicles (mean ± SEM) of the Antarctic minke whales
ComponentsnLactic acid (mg/dl)
Progesterone (ng/ml)
Estradiol-17β (ng/ml)<5

Follicular size (mm)
5–10

>10

Umbilical serum

18*1
136.0 ± 16.1a
13.3 ± 3.1
14.3 ± 2.6a25*1
96.8 ± 9.0a,b
16.7 ± 2.5
22.9 ± 4.0a,b

25*1
85.3 ± 10.0b
19.6 ± 3.9
34.6 ± 7.0 b

4*2
360.0 ± 46.3c
12.4 ± 4.8
1.2 ± 0.2 a,b*1, *2 See the footnotes of Table 2. a, b, c The different superscripts indicate significant differences (P<0.05).

with a significant difference between the small andlarge follicles (14.3 ± 2.6 and 34.6 ± 7.0 ng/ml,
respectively: P<0.05), and tended to be higher thanthat of the umbilical serum.

Discussion
The present study shows that the osmolarity of
the wFF of the small follicles was significantly low(363 mOsmol) compared with that in the medium
follicles, and that the osmolarity of the wFF of themedium and large follicles were similar to that of
the umbilical serum (380 to 390 mOsmol). Our previous reports [1, 3] showed that the nuclear
maturation rate of Antarctic minke whale oocytes
was approximately 30% after 96–120 h of IVM culture. In these studies, 15–20% fetal whale serum
was incorporated into IVM media with an osmolarity of approximately 310 mOsmol. Iwayama et al.
[4, 27] added 10 or 50% wFF into IVM media for

fresh or vitrified Antarctic minke whale oocytes,
and found that the IVM duration could be greatly
shortened to 30–40 h. The osmolarity of wFF (387.9± 3.1 mOsmol; n=23, [4]) is higher than those ofbovine and porcine FF (approximately 300 mOsmol: unpublished data, Iwayama H.). Addition of
10% wFF to IVM medium adjusting the osmolarity
to 390 mOsmol provides a better and more physiological environment for maturation of Antarctic
minke whale oocytes in vitro [4, 5, 27].
Although no significant difference was found in
the diameters of the zona pellucida intact oocytes
recovered from the three sizes of follicles, the diameters of the ooplasm of the oocytes recovered fromthe small and medium follicles were significantly
smaller than that recovered from the large follicles.
Formation of the vitelline space and shrinkage ofthe cytoplasm were not observed in any of theoocytes categorized into the three groups. This was
because there were significant differences in diameter of the ooplasm of the oocytes, but no difference

1270

NAGAI et al.

in diameter of the whole oocyte or thickness of thezona-pellucida; this was not clarified in the present
study. However, it should be kept in mind that thewhale oocytes used for diameter measurement
were derived from COCs vitrified immediately
after release from the wFF and had been placed in
vitrification solution with an unsuitable osmolarity(around 270 mOsmol) that did not contain wFF;
however, the present vitrification procedures may
not have only affected the ooplasm diameter. An
increase in osmolarity (up to 390 mOsmol) by adding wFF to a vitrification solution could be analternative means of maintaining oocyte quality
leading to subsequent IVM competence.
It has been well demonstrated that oocytes recovered from smaller follicles have less competency tofertilize and develop after maturation [13, 14, 19–22]. In the present study, the E2 concentration of
wFF collected from the small follicles was significantly or tended to be lower than those of wFFcollected from the two larger follicles. Iga et al. [25]previously reported that the E2 levels were detectable (≥0.2 ng/ml) in most follicles of Antarctic
minke whales and that large follicles (>8 mm indiameter) could be classified into growing andatretic follicles based on their E 2 levels. In thebovine, there is an increase in the concentration of
E2, as follicular size increases and there is no relationship between follicular size and theconcentration of P4 in the FF [28]. The concentrations of E2 and P4 in the wFF were similar to thoseseen in bovine follicles. Therefore, the present
results suggest that the oocytes and wFF derived
from large follicles with >10 mm in diametershould be used for IVM culture of whale oocytes.
All the Antarctic minke whales used in thepresent study were in the second trimester of gestation period, and the sample size (n=7 animals) wasvery limited. Therefore, we could not directly compare the metabolic components and electrolytes in
the FF with other species of whales and dolphins.
However, to the best of our knowledge, this is thefirst study of the concentrations of various components of FF, including steroid hormones, in relation
to follicular size in Antarctic minke whales. The
concentrations of various components of the wFFcollected from the medium and large follicles weresimilar, without any significant differences,although some of the characteristics of the wFF
were notable when compared with the umbilicalserum (Table 3). The concentrations of several

components (TP, GL, ALB, Na and Cl) were significantly higher than those in the umbilical serum,
whereas the T-CHO and Ca concentrations werelower than those in the umbilical serum. However,no significant differences were found in the concentrations of the chemical components among thethree groups of follicles, except for the E2 and lactic
acid concentrations. The concentration of lactic
acid of the small follicles was significantly higher
than those of the wFF collected from the two larger
follicle size groups. In the bovine, Iwata et al. [29]found that the concentration of lactic acid was negatively correlated with the GL (P<0.05) and Mg (–0.23, not significantly) concentrations of the FF.
However, in the present study, the GL concentrations of the small follicles (<5 mm) were notmeasured due to the limited volume of wFF. However, the concentrations of GL and T-CHO in
Antarctic minke whale FF were greater comparedwith those of bovine FF [29–31], and the concentrations of ALB, Ca and Mg were similar to those ofbovine FF. It may be that the characteristics of FFchange greatly during different developmental andestrous stages [32, 33]. These previous studies of
the characteristics in FF of domestic animals indicate that the concentrations of the FF components
are different depending on follicular size and suggested that the FF collected from larger follicles
positively affects oocyte quality and developmental
competence of oocytes after IVM culture [13, 14, 19–22]. The present wFF collected from the medium
and large follicles had higher concentrations of TP,
GL, ALB, Na, Cl and E2 and lower concentrations of
T-CHO, Ca and lactic acid than those of the umbilical serum. These data indicate that wFF would be amore suitable additive for IVM medium of Antarctic minke whale oocytes rather than umbilicalserum.
To produce whale embryos in vitro, we have been
attempted IVM, IVF and intracytoplasmic sperm
injection (ICSI) and IVC [1–5] and have obtained
early morula stage minke whale embryos; however, no blastocysts have been obtained to date.
The present results suggest that it is better to use
the follicular fluid recovered from larger follicles(>10 mm in diameter) for IVM culture and that thediameter of the ooplasm, osmolarity and concentrations of various components, including steroid
hormones of wFF should be considered. Thisapproach would be applicable to development of a
new IVM medium for Antarctic minke oocytes andFOLLICLE SIZES OF ANTARCTIC MINKE WHALE

could contribute to improvement of in vitro production of whale embryos in the near future. In
conclusion, the present results offer new information concerning the follicular components ofAntarctic minke whales and contribute whale
reproductive physiology, especially for improvement of IVM and related technologies in whales.

1271

Acknowledgements
The authors thank the captain and crew membersof the research base ship, “Nisshin-Maru”, for their
help in obtaining the Antarctic minke whale
ovaries.

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