http:// www.jstage.jst.go.jp / browse / jpsa
doi:10.2141/ jpsa.0120063
Copyright Ⓒ 2013, Japan Poultry Science Association.
Dibutoxybutane Suppresses Protein Degradation and Promotes Growth
in Cultured Chicken Muscle Cells
Tomomi Kamizono, Akira Ohtsuka, Fumio Hashimoto and Kunioki Hayashi
Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan
Dibutoxybutane (DBB) is a possible growth promoter contained in shochu distillery by-product. In the present
study, we synthesized DBB in order to confirm the growth promoting action. After determining the chemical
structure of the synthetic product by nuclear magnetic resonance and gas chromatography mass spectrometry, an
experiment was conducted using chicken skeletal muscle cells. DBB was mixed with the culture medium at the level
of 0, 0.015, 0.15, 1.5, 15 and 150 μg/ml and cells derived from thigh muscle of 13-day-old chick embryos were raised
for 6 days. The muscle cell growth was accelerated and the rate of protein degradation estimated from N τ-methylhistidine release into the medium was decreased by DBB. Furthermore, the mRNAs of muscle-specific E3 ubiquitin
ligases, atrogin-1/MAFbx and MuRF1, were decreased by DBB. However, mRNAs of ubiquitin, proteasome C2
subunit and μ-calpain were not affected by DBB. Activities of chicken’s two ubiquitous μ- and μ/m-calpain which
were quantified by casein zymography were decreased by DBB. These results show that DBB promotes muscle
growth due to a decrease in the rate of protein degradation.
Key words: dibutoxybutane, muscle growth, protein degradation
J. Poult. Sci., 50: 37-43, 2013
Introduction
For the better productivity of farm animals, increasing
edible meat without impairing their health and increasing the
production cost is important. Growth of skeletal muscle
takes place either by an increase in the rate of skeletal muscle
protein synthesis or by a reduction in the rate of the protein
degradation (Hayashi et al., 1985). Skeletal muscle contains
four proteolysis systems, the lysosomal, caspase, calpain and
ubiquitin-proteasome systems, and Goll et al. (2008) have
reported that ubiquitin-proteasome system and calpains are
responsible for turnover of myofibrillar protein which is the
major protein fraction in skeletal muscle.
The shochu distillery by-product (SDBP) is a functional
feedstuff due to its effect on skeletal muscle growth. SDBP
contains high amount of protein, vitamins E and C in addition
to growth promoters (Mahfudz et al., 1996a, b, 1997). It is
also the merit that it is very acidic because citric acid is produced during the fermentation.
Butoxybutyl alcohol (BBA) may be one of growth promoters reported by Mahfudz et al. (1997). It may be produced during fermentation of shochu, a traditional Japanese
Received: May 5, 2012, Accepted: July 3, 2012
Released Online Advance Publication: August 25, 2012
Correspondence: Dr. K. Hayashi, Department of Biochemical Science and
Biotechnology, Faculty of Agriculture, Kagoshima University, Kagoshima
890-0065, Japan. (E-mail: [email protected])
liquor, and the chemical structure was estimated to be BBA
by nuclear magnetic resonance (NMR) (Ohtani, Ohtsuka and
Hayashi, unpublished data). In the present study, we tried to
synthesize BBA to confirm the structure, but resulted compound was determined to be dibutoxybutane (DBB) an acetal. In the synthetic condition, BBA a hemiacetal might be
converted to DBB. DBB is another growth promoter reported by Mahfudz et al. (1997).
Although it had long been believed that the alcoholic fermentation by-products contain growth promoting factors, no
one was successful to extract and purify the factor. We
showed that feeding hexane extracts of SDBP decreased
expressions of genes relating to proteolysis, resulting in the
decreased rate of skeletal muscle protein degradation and the
increased muscle mass in chickens (Kamizono et al., 2010).
For the alcoholic fermentation of shochu, fungus such as
Aspergillus awamori is used for saccharification of raw
materials. High-performance liquid chromatography (HPLC)
analysis showed that the hexane extracts of SDBP contain 2
growth promoting factors DBB and BBA (Kamizono,
Ohtsuka and Hayashi, unpublished data) as reported by
Mahfudz et al. (1997). Recently, it was suggested that these
substances may be produced by Aspergillus awamori (Saleh
et al., 2011).
As we were successful in synthesizing DBB, we conducted
an in vitro experiment using chicken muscle cells to confirm
the growth promoting action of DBB. Chemical structure of
Journal of Poultry Science, 50 (1)
38
the synthesized DBB was confirmed by NMR and gas chromatography mass spectrometry (GC-MS).
Materials and Methods
Chemical Synthesis of DBB and Confirmation of Chemical
Structure
Five milliliters of butyl alcohol and 5 ml of butyl aldehyde
were reacted at room temperature with 0.01 g of p-toluenesulfonic acid as a catalyst. After 24 h, DBB was separated by
a Sephadex LH-20 column (35×700 mm) with a mobile
phase of methanol, dichloroethane and water (9:2:1). The
flow rate was 1 ml/min and DBB was monitored by absorbance at 275 nm using TOSOH UV-8010 (TOSOH, Tokyo,
Japan) and tricorder (EYELA, Tokyo, Japan). The fractions
of DBB were collected and the mobile phase was evaporated
by a vacuum evaporator. About 4 g of liquid (DBB) was obtained. The retention time of the synthesized DBB on HPLC
under the conditions previously reported (Mahfudz et al.,
1997) was the same as that of DBB extracted from SDBP.
The 1H- and 13C-NMR spectra were recorded with a NMR
spectrometer (JEOL JNM-ECA 600, JEOL, Japan, at the
Venture Business Laboratory in Kagoshima University) and
chemical shifts were expressed on a δ (ppm) scale with
tetramethylsilane (TMS) as an internal standard. The solvent
for NMR measurements was used with methanol-d4 (CD3
OD)＋0.03% TMS. The 2D field gradient (FG)-heteronuclear multiple quantum correlation (FG-HMQC) and the heteronuclear multiple bond correlation (HMBC) as well as
the 1H-1H total correlation (TOCSY) spectra were measured
to investigate the linkages among the protons and the carbons
(data not shown). Furthermore, the molecular weight of this
synthetic compound was determined by GC-MS (Polaris Q,
Thermo Finnigan, Tokyo, Japan) equipped with a capillary
column (DB-WAXetr, 60 m length, 0.25 mm I.D., 0.25 μm
film-thickness, J&W Scientific Inc., CA, USA). The synthesized DBB was identified in its chemical structure as shown
in Fig. 1.
Cell Cultures
The cells were isolated from the thigh muscle of 13-dayold chick embryos (Mahfudz et al., 1997; Nakashima et al.,
1998) and kept in liquid nitrogen until used for cell culture.
The frozen muscle tissue was quickly thawed and digested
with dispase (2,000 U/ml) for 10 min at 37℃. The cell suspension was then passed through a net and centrifuged 180×
g for 5 min. The supernatant was aspirated, and the cell
pellet was dispersed into basal medium, M-199 containing
15% calf serum, 2.5% chicken embryo extract, streptomycin
Fig. 1.
The structure of DBB.
(100 mg/l) and penicillin (105 U/l). The cell suspension was
transferred to a 35-mm uncoated culture dish to allow fibroblast attachment. After 40 min, the unattached cells were
recovered and transferred to another uncoated dish. This
adhesion method was repeated three times. The cell numbers
were counted and plated onto gelatin-coated 6-well plates at
a density of 2.5×105 cells/well. The cells were grown at
37℃ in a 5% CO2 -enriched atmosphere of humidified air.
After 24 h, the medium was replaced with a medium containing different concentrations of DBB. Levels of DBB were
0.015, 0.15, 1.5, 15 and 150 μg/ml dissolved in ethanol (final
concentration was 0.1% of medium). Control medium was
included same amount of ethanol.
Measurement of Protein Content and N τ -methylhistidine
Release
The protein content was determined by the Bradford
method with bovine serum albumin as standard (Bradford,
1976). Protein degradation was evaluated by measuring N τmethylhistidine release into the medium as described previously (Hayashi et al., 1987; Kamizono et al., 2010). Culture medium was mixed with 20% sulfosalicylic acid and
centrifuged at 9,600×g for 5 min. The supernatant was recovered and evaporated under reduced pressure. The residue
was dissolved in 0.2 M pyridine and applied to a cationexchange column (7×60 mm, Dowex 50W-X8, 200-400
mesh, pyridine form). After most of the acidic and neutral
amino acid were washed out with 0.2 M pyridine, N τ methylhistidine was eluted with 1 M pyridine and collected.
The solvent was evaporated and the residue was dissolved in
mobile phase (15 mM sodium 1-octanesulfonate in 20 mM
KH2 PO4). An aliquot was injected into a HPLC (LC-6A,
Shimadzu, Kyoto, Japan) equipped with an Inertsil ODS80A column (4.6×250 mm, 5 μm, GL Sciences, Tokyo, Japan). The column was attached to an oven at 50℃. A fluoromonitor (RF-535, Shimadzu, Kyoto, Japan) set at an excitation wavelength of 365 nm, and an emission wavelength
of 460 nm was used to monitor the fluorescence derived from
the reaction with o-phthalaldehyde.
Measurement of mRNA Levels
The mRNA levels were determined by real-time PCR, as
described previously (Kamizono et al., 2010). Total RNA
was extracted from muscle cell mono layer using an RNeasy®
Fibrous Tissue Mini Kit (QIAGEN, Tokyo, Japan), according to the manufacturer’s protocol. The RNA concentration
and purity were determined spectrophotometrically using
A260 and A280 values in a photometer (BioPhotometer, Eppendorf, Hamburg, Germany). The ratio of A260/A280 of all
samples was between 1.8 and 2.0. Complementary DNA
was synthesized 800 ng RNA per 20 μl reaction solution with
PrimeScript® RT reagent Kit (Perfect Real Time, TaKaRa,
Shiga, Japan) by Program Temp Control System PC320
(ASTEC, Fukuoka, Japan), which was set as reverse transcription: 37℃ for 15 min, inactivation of reverse transcriptase: 85℃ for 5 s and refrigeration: 4℃ for 5 min. Gene expression was measured by real-time PCR using 7300 Real
Time PCR system (Applied Biosystems, Foster City, CA,
USA) with SYBR® Premix Ex TaqTM (Perfect Real Time,
Kamizono et al.: Function of DBB as Growth Promoter
TaKaRa, Shiga, Japan). Thermal cycle is as follows: 1 cycle
95℃ for 10 s, 60 cycles at 95℃ for 5 s and 60℃ for 31 s.
Primers were referred to literatures (Nakashima et al., 2005,
2009; Tesseraud et al., 2009). Expression of glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an
internal standard and was not significantly different among
the experimental groups. The mRNA levels in the control
were arbitrarily set to 1.0.
Measurement of Calpain Activity
Casein zymography for measurement of the calpain activity was performed according to the protocol described by
Lee et al. (2007) with slight modification. For sample preparation, muscle cell mono layer was collected with extraction buffer (50 mM Tris-HCl, pH 8.3, 20 mM EDTA, 10 mM
EGTA and 0.1% β-mercaptoethanol). Then, collected sample was repeated 3 times of freeze-thaw cycles to crush cell
layer with gentle condition. Protein content was measured
by above-mentioned method and prepared sample solution
was added to sample buffer (150 mM Tris-HCl, pH 6.8, 20%
glycerol, 0.75% β-mercaptoethanol and 0.04% bromophenol
blue) at a 4:1 ratio. Samples (10-20 μg of protein) were
loaded wells of casein mini-gels composed of resolving gels
(0.2% casein, 10% acrylamide, 0.4% bisacrylamide, 0.04%
ammonium persulphate and 0.28% TEMED in 375 mM TrisHCl, pH 8.8) and stacking gels (4% acrylamide, 0.16%
bisacrylamide, 0.04% ammonium persulphate and 0.28%
TEMED in 330 mM Tris-HCl, pH 6.8) and electrophoresed
at 100 V for 4 h at 4℃ in running buffer (25 mM Tris-HCl,
pH 8.3, 192 mM glycine, 1 mM EDTA, 1 mM EGTA and 1
mM DTT). After electrophoresis, the gels were rinsed 2
times for 30 min at 20℃ with slow shaking in activation
buffer (20 mM Tris-HCl, pH 7.5, 3 mM CaCl2), and then
they were incubated for 24 h at 20℃ in the activation buffer
plus 10 mM DTT. Finally, the gels were strained for 1 h with
Coomassie brilliant blue R-250 and then destained overnight
(18 h) in decoloration buffer (5% methanol and 8% acetic
acid). Calpain activity appears as white bands on a blue
background due to digestion of casein. The resulting signals
were quantified using Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Lee et al. (2007) reported that the signal was linear between 5 and 50 μg of
protein extract from the pectoralis major muscle, loaded on a
gel. All the assays were performed in the presence of 10-20
μg of protein content giving a signal in the range of linearity.
The signals were corrected by protein content loaded on each
lane and the calpain activity in the control was arbitrarily set
to 1.0.
Statistical Analysis
Values are presented as means±standard error (SE). The
significance of difference was evaluated by ANOVA and
Tukey’s multiple-range test. All statistical analysis was performed with the general linear model procedures of SAS
(version 9.2, SAS Institute). A p value ＜ 0.05 was considered statistically significant.
39
Results
Determination of Chemical Structure of DBB
We synthesized DBB and the chemical structure was
determined (Fig. 1). The structure was confirmed as DBB
by 1H-NMR and 13C-NMR. The NMR results were shown
below. 1 H-NMR (CD3 OD) δ: 0. 92 (3H, t, J＝6. 8 Hz, 4CH3), 0.93 (6H, t, J＝7.5 Hz, 4’-CH3×2), 1.37 (2H, m, 3CH2), 1.39 (4H, m, 3’-CH2×2), 1.54 (4H, m, 2’-CH2×2),
1.56 (2H, m, 2-CH2), 3.42 (2H, ddd, J＝9.5, 6.8, 6.1 Hz,
1’-HA), 3. 59 (2H, ddd, J＝9. 5, 6. 8, 6. 1 Hz, 1’ -HB), 4. 46
(1H, t, J＝5.4 Hz, 1-H). 13 C-NMR (OD3 OD) δ: 14.2 (4’CH3×2), 14. 3 (4-CH3), 19. 0 (3-CH2), 20. 4 (3’ -CH2×2),
33.1 (2’-CH2×2), 36.8 (2-CH2), 66.6 (1’-CH2O×2), 104.5
(1-ketal-C). EI-MS (by GC-MS) m/z 202 [M]+ (calcd for
C12 H26 O2, 202.19), 159 [M-C3 H7]+, 129 [M-C4 H9 O]+, 73
[C4H9O]+, 57 [C4H9]+.
According to the HMQC spectrum (1H, 13C-NMR, δ ppm),
there observed a set of two methyl groups (0.93, 14.2), a
methyl group (0.92, 14.3), two sets of two methylene groups
(1.39, 20.4 and 1.54, 33.1), two methylene groups (1.37,
19.0 and 1.56, 36.8), a set of carbinol methylene (3.42 and
3.59 with 66.6), and a ketal methine (4.46, 104.5). The
numbers of protons are confirmed by the integration of 1HNMR assignment. In the HMBC spectrum, the two methylene protons (1.37) correlated with a methylene carbon (36.8)
and the ketal carbon (104.5) and the two methylene protons
(1.56) showed correlations with the methylene carbon (19.0)
and the ketal carbon (104.5) while the methyl three protons
(0.92) correlated with the two methylene carbons (19.0 and
36.8), thus these signals are unambiguously arising from the
structure of a n-butyl group with a ketal sequence (4.46,
104.5). On the other hand, seemingly the three protons of the
methyl group (0.93) showed the correlations with the two
methylene carbons (20.4 and 33.1) and seemingly the two
protons of the two methylene groups (1.39 and 1.54) showed
the correlations with carbons (14.2, 33.1 and 66.6, and 14.2,
20.4 and 66.6, respectively). The carbinol methine protons
(3.42 and 3.59) also correlated with seemingly the two methylene carbons (20.4 and 33.1), thus these signals are suggested to be from the other n-butyl alcohol group. Since the
integration of 1H-NMR spectrum showed the presence of two
sets of the n-butyl alcohol groups, it is presumed that there is
asset of two n-butyl alcohol groups. Furthermore, the carbinol methine protons (3.42 and 3.59) showed the long range
correlation with the ketal methine (104.5) and the ketal proton (4.46) also showed the long range correlation with the
carbinol methylene carbon (66.6). Based on these observations, the two n-butyl alcohol groups are attached to the nbutyl alcohol with a ketal sequence (as the two butoxy alcohol groups), thus the molecule has a symmetric structure.
The mass spectrum supported this presumed structure; the
fragment ion peak where a propyl group is detached from the
molecule was observed at 159 [M-C3H7]+ and the fragment
ion peak where a butoxy group is detached from the molecule
was observed at 129 [M-C4H9O]+. Based on all the spectral
data, the structure of the compound was concluded to be
40
Journal of Poultry Science, 50 (1)
Effect of DBB on Expressions of Proteolysis Related Genes
in Cultured Chicken Muscle Cells
We examined the effect of DBB on expressions of genes
related to muscle proteolysis since it was suggested that DBB
suppresses skeletal muscle protein degradation. Atrogin-1/
MAFbx and MuRF1 were selected as ubiquitin ligases.
Atrogin-1/MAFbx mRNA was significantly decreased by
DBB (p＝0.001: ANOVA, Fig. 3A). MuRF1 mRNA was also
decreased by DBB (p＝0.046: ANOVA, Fig. 3B). However,
ubiquitin, proteasome C2 subunit and μ-calpain mRNAs
were not affected by DBB (Fig. 3C-E). These results suggest that reduction of the rate of proteolysis in skeletal
muscle is attributed to suppression of atrogin-1/MAFbx and
MuRF1 mRNAs expressions.
Effect of DBB on Activities of μ- and μ/m-Calpain in Cultured Chicken Muscle Cells
In Fig. 4A, it showed that in each lane, 2 bands existed,
denoting μ-calpain (upper) and μ/m-calpain (lower), respectively. Although mRNA of μ-calpain was not affected by
DBB (Fig. 3E), activities of chicken’s two ubiquitous calpains, μ- and μ/m-calpain were influenced (Fig. 4B-C). μCalpain activity was significantly decreased by DBB (p＝
0.020: ANOVA, Fig. 4B). In addition, μ/m-calpain activity
was also decreased by DBB (p＝0.001: ANOVA, Fig. 4C).
These results suggest that DBB directly reduces calpain
activity without suppressing of calpain mRNA expression.
Discussion
Effect of DBB on protein content (A) and N τmethylhistidine release (B) in cultured chicken muscle
cells. Data represent means±SE (n＝6). Means with
different letters are significantly different at p＜0.05.
Fig. 2.
DBB (1, 1-di-n-butoxy-n-butane). DBB is presumed to be
formed by an addition of n-butyl alcohol to BBA (1-nbutoxy-1-hydroxy-n-butane).
Effect of DBB on Growth and Protein Degradation in Cultured Chicken Muscle Cells
The effect of DBB on growth was evaluated by total protein content and the proteolytic effect was evaluated by N τmethylhistidine release into the medium. Protein content
was significantly increased by DBB at levels from 0.15 to
150 μg/ml (Fig. 2A). But no difference was observed between control and the group of 0.015 μg/ml (Fig. 2A). The
N τ-methylhistidine release into the medium was significantly
suppressed by DBB at levels of 0.15 and 150 μg/ml, and
tended to be decreased at levels of 1.5 and 15 μg/ml compared with control, but no effect was observed at the level of
0. 015 μg/ml (Fig. 2B). When these results were analyzed
ANOVA, significant differences of both of protein content
and N τ -methylhistidine release into the medium were observed by DBB treatment (p＝0.001 and p＝0.005, respectively). These results suggest that DBB suppresses muscle
protein degradation and promotes growth of muscle cells.
Although alcohol fermentation by-products have been
thought to contain growth-promoting substances, none of
these substances has been identified by any study to date. In
the previous studies, SDBP has been focused as feedstuff
showing growth promotion (Mahfudz et al., 1996b). However, the mechanism of the growth promoting effect is still
unclear. The SDBP contains not only growth promoting substance but also many useful nutrients such as protein and
vitamins. Recently, we found DBB and BBA as possible
growth promoting substances from SDBP. Then, we synthesized DBB chemically while synthesizing BBA was not
capable at the present time. Chemical structure of the synthesized DBB was confirmed by NMR and GC-MS (Fig. 1).
Previously, we reported that hexane extracts of SDBP
decreased gene expressions relating to calpain and ubiquitinproteasome system and reduced the rate of skeletal muscle
proteolysis in chickens (Kamizono et al., 2010). We confirmed that the extracts contain DBB. From these results, we
thought that DBB is responsible for the effects on skeletal
muscle growth and proteolysis. In order to prove the growth
promoting effect of DBB, the present in vitro experiment was
conducted using cultured chicken muscle cells.
In the present experiment, DBB increased protein content
and decreased N τ -methylhistidine release (Fig. 2). N τ Methylhistidine is a component of skeletal muscle protein,
actin and myosin (Asatoor and Armstrong, 1967). When
skeletal muscle protein is degraded, N τ -methylhistidine released and excreted in the urine, plasma and medium, and be
not reused for protein synthesis due to the lack of existing N τ
Kamizono et al.: Function of DBB as Growth Promoter
Effect of DBB on mRNA levels of atrogin-1/MAFbx (A),
MuRF1 (B), ubiquitin (C), proteasome C2 subunit (D) and μ-calpin
(E) in cultured chicken muscle cells. Data represent means±SE (n＝
5-6). Means with different letters are significantly different at p＜0.05.
Fig. 3.
41
Journal of Poultry Science, 50 (1)
42
Effect of DBB on casein zymography of calpains (A) and
activity of μ-calpain (B) and μ/m-calpain (C) in cultured chicken
muscle cells. Data represent means±SE (n＝5-6). Means with different letters are significantly different at p＜0.05.
Fig. 4.
-methylhistidine tRNA (Young et al., 1972). Therefore, N τmethylhistidine release from the muscle cells was used as an
index of the rate of myofibrillar protein degradation in the
present study. These results indicate that increase in protein
content by DBB was due from the reduction in skeletal muscle proteolysis.
In skeletal muscles, ubiquitin-proteasome system and calpains are thought to play major roles in protein degradation
(Goll et al., 2008). The ubiquitin-proteasome system is an
ATP-dependent proteolysis system that requires polyubiquitination of the target protein as a marker of degradation by
the 26S proteasome (Ciechanover, 2006). Polyubiquitination of the target protein consists of the covalent linkage of
ubiquitin molecules to one or more lysine residues of a pro-
tein. The process involves the 3 types of ubiquitination enzymes E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzymes) and E3 (ubiquitin ligases), and E3 provides specificity to the ubiquitin-proteasome system (Mearini
et al., 2008). It has also been demonstrated that the ubiquitin-proteasome system is controlled by the expression of 2
important E3 ubiquitin ligases such as atrogin-1/MAFbx and
MuRF1 (Franch and Price, 2005; Glass, 2005; Szewczyk and
Jacobson, 2005). Thus we measured the mRNAs of atrogin1/MAFbx and MuRF1 as muscle proteolysis markers.
Expressions of atrogin-1/MAFbx and MuRF1 mRNAs were
significantly decreased by DBB (Fig. 3A-B). We were also
measured expressions of components of ubiquitin-proteasome system, including ubiquitin and proteasome C2 subunit
Kamizono et al.: Function of DBB as Growth Promoter
mRNAs, however, these were not affected by DBB (Fig. 3CD).
Calpains are a family of intracellular Ca2+-dependent cysteine proteases that are ubiquitously expressed in many cells
and tissues (Suzuki et al., 1995). In the previous study, we
reported that hexane extracts of SDBP containing DBB
suppressed gene expression of μ-calpain (Kamizono et al.,
2010). Calpains may play a significant role in initiation of
muscle protein degradation by releasing protein fragments
for proteolysis of the ubiquitin-proteasome system. Smith
and Dodd (2007) have reported that calpain activation inhibits the Akt signaling pathway. Interestingly, DBB reduced enzyme activity (Fig. 4B-C) but not gene expression
(Fig. 3E) of calpain, and thus activity of Akt and their downstream factor may be influenced followed by decreasing in
skeletal muscle proteolysis.
In conclusion, the present study supports the idea that
DBB suppresses proteolysis and promotes growth in skeletal
muscle.
Acknowledgments
This study was supported by the Japan Poultry Science
Association for a providing a travel grant to allow presentation at 9th Asia Pacific Poultry Conference in Taiwan. We
are grateful to Kagoshima Chicken Foods Co., Ltd., (Kagoshima, Japan) for a supply of fertile eggs.
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