Research Article

Received: 24 February 2013 Revised: 16 June 2013 Accepted article published: 20 September 2013 Published online in Wiley Online Library: 21 October 2013

(wileyonlinelibrary.com) DOI 10.1002/jsfa.6416

Comparison of postmortem proteolysis

between Pekin and Muscovy duck breast
muscles
Chien-Chang Liao and Rong-Ghi R Chou

Abstract:
BACKGROUND: Duck muscle is a popular source of red meat in Asia. However, information regarding the postmortem proteolysis
of skeletal muscle between duck species is very limited. Therefore, the purpose of this study was to compare the postmortem
calpain and desmin degradations between Pekin (PD) and Muscovy (MD) duck breast muscles stored at 5 C.

RESULTS: The pH and /m-calpain activity were not different (P > 0.05) between PD and MD postmortem muscles. However,
-calpain activity and desmin content decreased more rapidly (P < 0.05) in PD than in MD samples.

CONCLUSION: Therefore, our results suggest that the postmortem proteolysis is more rapid and extensive in breast muscle
from PD compared to MD.
c 2013 Society of Chemical Industry

Keywords: duck skeletal muscle; postmortem proteolysis; calpain; desmin

INTRODUCTION therefore, was to compare the postmortem calpain and desmin

Changes in postmortem skeletal muscle stored at refrigerated
temperature have been studied for years. It has been established
that degradation of myofibrillarcytoskeletal proteins is a natural
process that occurs during postmortem conversion of muscle
to meat and continues during storage.1,2 Calcium-dependent
proteases (calpains) are generally thought to be responsible
for this degradation3 although the precise mechanism is not
well-understood. There are two ubiquitous calpains, - and
/m-calpain, that are present in avian skeletal muscle cells.4,5 It
was reported that both calpains were activated and autolysed
in postmortem chicken muscle.6 Recent studies7 reported that
-calpain, rather than /m-calpain, activity was correlated
with postmortem degradation of desmin and troponin-T in
duck muscle, supporting the essential role of -calpain in the
postmortem proteolysis of skeletal muscles.8
It has been reported that the postmortem degradation
could be influenced by a variety of factors.9 Among those
factors, the degradation of postmortem skeletal muscle from
various mammalian species was frequently compared. Previously,
Koohmaraie et al.10 compared postmortem desmin degradation
in pork, beef and lamb longissimus muscles and reported that the
most extensive desmin degradation was found in pork muscle.
Recently, Soltanizadeh et al.11 showed that postmortem troponin-
T degradation and appearance of a 30 kDa band were more rapid
in camel than in bovine semi-tendinosus muscle. On the other
hand, among various duck species, Pekin (Anas platyrhynchos)
and Muscovy (Cairina moschata) duck skeletal muscles are very
popular sources of red meat in Asia. However, differences in the
postmortem degradation of skeletal muscle between these two
2846
changes between Pekin and Muscovy duck skeletal muscles stored
at 5 C.
MATERIALS AND METHODS
Sample preparation
The Institutional Animal Care and Use Committee, National Chiayi
University, approved the use of animals in this study. Mature (13-
to 14 month-old) male Pekin (PD, an average live weight of approx.
4.3 kg) and Muscovy ducks (MD, an average live weight of approx.
4.2 kg) were obtained from the poultry farm of National Chiayi
University and used in this experiment. After resting overnight
with sufficient drinking water at 2025 C, six PD and six MD
ducks were harvested on same day in a local abattoir according
to a standard commercial practice. Both sides of breast muscles
(Pectoralis superficialis) were excised from the carcasses at 1 h
postmortem. The breast muscle from each duck was cut into five
equal portions and randomly allocated to five sampling times.
The portions were then vacuum-packed individually and stored at
5 C for 0 (1 h postmortem), 1, 3, 7 and 14 days. At the end of
each storage period, the samples were quickly cut with a scalpel,
immediately snap-frozen in liquid nitrogen and stored at 80 C
until required for subsequent analysis. Each analysis was done
twice on samples from each duck at each storage time.
Correspondence to: Rong-Ghi R Chou, Department of Animal Science, National
Chiayi University, 300 University Road, Chiayi City, 60083 Taiwan. E-mail:
chourg@mail.ncyu.edu.tw

species have not been examined so far. The purpose of this study, Department of Animal Science, National Chiayi University, Chiayi City, Taiwan

J Sci Food Agric 2014; 94: 28462849 www.soci.org 

c 2013 Society of Chemical Industry
Postmortem proteolysis of PD and MD muscle
pH measurement
A 5 g aliquot from each duck sample was crushed in liquid nitrogen
for the pH determination by the method of Farouk and Swan.12
The pH for each sample was measured by a Suntex 470 pH meter
(Suntex Instruments Co., Taiwan) with a glass electrode.
Casein zymography
The procedure used for protein extraction was based on the
method of Veiseth et al.13 Briefly, 5 g of each duck sample was
homogenised in three volumes of extraction buffer [100 mmol L1
Tris base, 10 mmol L1 EDTA, 0.5 g kg1 2-mercaptoethanol
(MCE), pH 8.3] at 5 C. Homogenates were centrifuged at
22 000 g for 25 min at 5 C. The protein concentration of
the supernatant was determined by using a modified biuret
method.14 Zymograms were routinely run in 100 g kg1 gels
(acrylamide:methylenebisacrylamide, 37.5: 1, w/w) containing 2.1
g kg1 casein (w/v) by the method of Raser et al.15 The sample
buffer [150 mmol L1 Tris-HCl, pH 6.8, 200 g kg1 glycerol, 0.5
g kg1 MCE, 0.2 g kg1 (w/v) bromophenol blue] was added to
the protein extract at a ratio of two parts of the buffer to three
parts of protein extract (v/v). The same amount of protein (250 g)
from each sample was loaded onto each well of the casein gels.
The casein minigels (0.75 mm; Bio-Rad Laboratories, Herculues,
CA, USA) were pre-run at 100 V for 15 min, 5 C, with a running
buffer containing 25 mmol L1 Tris-HCl, 0.5 g kg1 MCE, 192 mmol
L1 glycine, and 1 mmol L1 EDTA (pH 8.3) before samples were
loaded onto the wells. The gels were run at 100 V for 2 h, 5 C,
and then incubated at room temperature in three changes of a 50
mmol L1 Tris-HCl (pH 7.5) buffer containing 0.5 g kg1 MCE and
4 mmol L1 CaCl2 with slow shaking for 1 h. This was followed by
16 h incubation in the same buffer at 37 C. The gels were then
stained for 2 h with Coomassie blue (R-350) and destained with
200 g kg1 methanol and 70 g kg1 acetic acid.
Western blot analysis
Breast myofibrils were isolated at 2 C by using differential
centrifugation according to the method of Goll et al.16 with the
modifications by Huff-Lonergan et al.17 Briefly, 10 g of each sample
were homogenised in 10 volumes of a standard salt solution buffer
(100 mmol L1 KCl, 20 mmol L1 potassium phosphate, pH 6.8, 2
mmol L1 MgCl2 , 2 mmol L1 EGTA, 1 mmol L1 NaN3 ). After the
second wash with 100 mmol L1 KCl solution and centrifugation
with 10 g kg1 Triton X-100 (v/v), 100 mmol L1 KCl, 2 mmol L1
MgCl2 , 1 mmol L1 EGTA, 1 mmol L1 NaN3 , and 20 mmol L1
potassium phosphate, pH 6.9, re-suspension of the myofibrils in
all succeeding steps was accomplished by vigorous stirring with
a small plastic spatula. Following the final wash with 100 mmol
L1 KC1, the myofibrils were washed twice by re-suspension in
10 volumes (w/v; weight in grams of the original ground muscle
sample) of 5 mmol L1 Tris-HC1, pH 8.0, and were centrifuged at
3020 g for 10 min. Following the second 5 mmol L1 Tris-HC1,
pH 8.0, wash, the myofibrils were re-suspended in four volumes
(w/v; weight in grams of the original ground muscle sample)
of 5 mmol L1 Tris-HC1, pH 8.0. The protein concentration of
myofibrils was determined by using a modified Biuret method.14
Myofibril samples for SDS-PAGE were prepared by the method
of Fritz and Greaser.18 The SDS-PAGE was performed in a 120 g
kg1 Tris-glycine slab gel (acrylamide:methylene bisacrylamide,
37.5: 1, w/w) by the method of Laemmli.19 The protein (150
g) from each sample was loaded onto each well. Molecular
J Sci Food Agric 2014; 94: 28462849 
c 2013 Society of Chemical Industry
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weight markers ranging from 4000 to 250 000 (SeeBlue Pre-
stained Standard, LC5625; Invitrogen Co., Carlsbad, CA, USA)
were used as protein standards. After electrophoresis, proteins
were transferred from a 120 g kg1 slab gel to a nitrocellulose
membrane by the method of Towbin et al.20 The membrane was
incubated in a 50 g kg1 bovine serum albuminphosphate buffer
solution (BSA-PBS) for 30 min at 37 C and then was washed three
times (5 min each) in a 1 g kg1 BSA-PBS solution at 25 C. A
monoclonal antibody to desmin (Clone DE-U-10, 1:500 dilution)
was used as a primary antibody. The membrane was incubated
with the primary antibody for 2 h at room temperature, washed
three times (5 min each) in 1 g kg1 BSA-PBS, incubated with
a secondary antibody, goat anti-mousehorseradish peroxidase
(HRP) for 2 h at room temperature, washed twice (5 min each) in
1 g kg1 BSA-PBS solution and twice (1 min each) in deionised
water. The colour was developed by using SIGMAFASTTM 3,3 -
diaminobenzidine tablets. The primary and secondary antibodies
as well as the SIGMAFASTTM 3,3 -diaminobenzidine tablets were
purchased from the SigmaAldrich Company (St. Louis, MO, USA).
Image analysis
Two representative casein gels and blots with all samples from
each duck (experimental unit) used for image analysis. The bands
in blots or casein gels, which were incubated in the presence of
4 mmol L1 Ca2+ to activate all calpain isoforms, were digitised
with a scanner (Model J131B; Epson) using Photoshop software.
Each blot or gel included a pooled 0-day Pekin duck sample
as a reference standard to normalise the band intensities. The
resulting signals were quantified using ImageJ (version 1.44i,
made by Wayne Rasband, National Institutes of Health, MD, USA).
The activities of -calpain and /m-calpain, as well as the desmin
contents in 1-, 3-, 7- and 14-day postmortem PD samples and at
0-, 1-, 3-, 7- and 14-day postmortem MD samples were determined
and compared to those in 0-day PD samples, respectively. The
results were expressed as percentages of the 0-day PD samples.
Statistical analysis
A split-plot design was used in this study. Whole units were
the breast muscles from each avian species and sub-units
were the muscle samples taken at each sampling time. All data
were analysed using the Mixed Model procedure of SAS (PROC
Mixed).21 The model included avian species, time postmortem and
their interaction (species time postmortem) for fixed effects and
birds for random effects. A Tukeys honestly significant difference
test was used to compare multiple means.
RESULTS AND DISCUSSION
The objective of this study was to compare the difference in
postmortem calpain and desmin degradation between Pekin (PD)
and Muscoy (MD) ducks. The ANOVA indicated that time was a
significant source of variance for all traits (Table 1). Species and
species time were significant sources of variance for -calpain
activity and presence of intact desmin.
Results shown in Fig. 1 indicated that breast muscle pH was
not different (P > 0.05) between PD and MD samples. However, pH
declined (P < 0.001) from 6.43 on day 0 to 5.96 on day 1 for sam-
ples from both types of duck. Breast muscle pH stabilised from days
1 to 14 as values from these sampling days, as well as between these
2847
days, were not different from each other (P > 0.05). These results
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 www.soci.org C-C Liao, R-G R Chou

125
Table 1. Analysis of variance of fixed effectsa

Relative -calpain activity, %

-Calpain /m-Calpain 100
Effect df pH activity activity Desmin

Species (S) 1 0.30 75.61 0.20 24.91 75

Time (T) 4 108.73 217.32 19.93 43.09
ST 4 0.74 28.32 0.78 6.98 50
a F-values.
< 0.05 < 0.01 < 0.001 25

0
0 2 4 6 8 10 12 14
7.0
Postmortem time, day

6.5 Figure 3. Postmortem changes in -calpain activity of Pekin (PD) and

Muscovy (MD) duck muscle samples stored at 5 C. Activity is expressed
pH

as the percentage of the 0-day PD -calpain activity, which was taken as

6.0
100%. SEM = 3.2%. Black squares, PD; white squares, MD.

5.5 125
0 2 4 6 8 10 12 14

Relative /m-calpain activity, %

Postmortem time, day
100
Figure 1. Postmortem changes in pH of Pekin (PD) and Muscovy (MD)
breast muscles stored at 5 C. Each point is the average of six replications
75
(n = 6). SEM = 0.065. Black squares, PD; white squares, MD.

50

25

0
0 2 4 6 8 10 12 14
Postmortem time, day
Figure 2. Zymograms showing, in the presence of 4 mmol L1 calcium,
postmortem changes in - and /m-calpain activities in PD (a) and MD (b) Figure 4. Postmortem changes in /m-calpain activity of Pekin (PD) and
duck samples stored at 5 C. Lane 1, 0-day samples; lane 2, 1-day samples; Muscovy (MD) duck samples stored at 5 C. Activity is expressed as the
lane 3, 3-day samples; lane 4, 7-day samples; lane 5, 14-day samples. percentage of the 0-day PD /m-calpain activity, which was taken as 100%.
= -calpain; /m = /m-calpain. SEM = 5.7%. Black squares, PD; white squares, MD.

indicated that the rate of postmortem pH decline measured at 1 h
postmortem was very similar in PD and MD muscles stored at 5 C.
In good agreement with a previous study,7 results of casein
zymograms presented in Fig. 2 showed that the upper and the
bottom rows of bands were - and /m-calpain bands in the
presence of 4 mmol L1 Ca2+ , respectively. The -calpain band
disappeared in 7-day and 14-day PD samples (Fig. 2a), but it
remained visible in 14-day MD samples (Fig. 2b). On the other
hand, the /m-calpain bands were clearly present in both PD and
MD samples during the entire 14 days of postmortem storage
(Fig. 2). However, a faint band just below the /m-calpain band
in 3-day PD and MD samples began to appear on the casein gels
(Fig. 2). Previous studies7 reported that this band is likely to be a
degradation product of /m-calpain in postmortem duck muscle.
When the zymograms in Fig. 2 were subjected to image analysis,
the results presented in Fig. 3 indicated that the -calpain activity in
0-day PD samples, which was taken as 100%, was higher (P = 0.017)
than that in 0-day MD samples, which was 84% of the 0-day PD
samples. As postmortem time proceeded, the -calpain activity
rapidly decreased (P < 0.0001) to 31% and to 14% of the 0-day
PD -calpain activity in 1-day and 3-day PD samples, respectively.
Subsequently, there was little -calpain activity in 7- and 14-
2848
activity was 84% of the 0-day -calpain activity in PD samples
(P = 0.017) and was reduced to 59% by day 1 (P < 0.0001). When
compared with the 0-day PD -calpain activity, the MD -calpain
was further reduced to 45% (P = 0.0139) in 3-day samples, to
35% (P = 0.541) in 7-day samples and to 22% (P = 0.152) in
14 day samples. However, the -calpain activity in the 3-day MD
samples was significantly (P = 0.0003) higher than that in the 14-
day MD samples. These results indicate that -calpain activation
and autolysis were more rapid and extensive in PD than in MD
samples.
Figure 4 shows there were almost identical changes in /m-
calpain activity in PD and MD samples rather than the distinct
differences in -calpain activity shown in Fig. 3. The /m-calpain
activity in 0-day PD samples, which was taken as 100%, and
MD samples, which was 98% of the 0-day PD samples, initially
reduced (P < 0.001) to 85 and 86% in 1-day PD and MD samples,
respectively, indicating that the /m-calpain activation and
autolysis had occur. The /m-calpain activity showed no further
decrease (P > 0.05) from day 1 to day 14 and reached 70% in
both PD and MD samples, suggesting a similar rate of /m-calpain
autolysis that had occurred in both duck muscles stored at 5 C.
Desmin, a calpain-sensitive protein, is very susceptible to

day PD samples. In MD samples, however, the 0-day -calpain degradation in skeletal muscles22,23 during postmortem storage.

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c 2013 Society of Chemical Industry J Sci Food Agric 2014; 94: 28462849
Postmortem proteolysis of PD and MD muscle
D- D-
(a)
125
Relative desmin content %
100
75
50
25
0
0 2 4 6 8 10
(c) Postmortem time, day
Figure 5. Western blot showing changes in desmin of Pekin (PD) (a) and
Muscovy (MD) (b) duck samples during postmortem storage at 5 C. Lane 1,
0-day samples; lane 2, 1-day samples; lane 3, 3-day samples; lane 4, 7-day
samples; lane 5, 14-day samples. D = Desmin. (c). Postmortem changes
in relative desmin amount in PD and MD duck samples stored at 5 C.
The relative desmin amount in the 0-day PD samples was taken as 100%.
SEM = 6.4%. Black squares, PD; white squares, MD.
Postmortem changes in desmin of both PD and MD samples stored
at 5 C were examined by western blotting (Fig. 5). Results shown
in Fig. 5 indicated that the desmin band in PD samples was clearly
visible from day 0 to day 3, became faintly visible at day 7 and
disappeared at day 14 (Fig. 5a). However, the desmin band was
clearly visible from day 0 to day 14 in MD samples (Fig. 5b). The
extent of desmin proteolysis was quantified as a percentage of the
relative content of the PD desmin band at day 0, which was taken
as 100%. As time postmortem proceeded (Fig. 5c), the relative
content of PD desmin was quickly reduced to 70% in 1-day
samples (P = 0.003) and to 43% in 3-day samples (P < 0.001).
The desmin content further decreased to 10% in 7-day samples
(P < 0.001) and then statistically unchanged to 5% in 14-day
samples (P = 0.996) (Fig. 5c). In MD samples, however, the 0-day
desmin content was 104% of the 0-day PD samples (P = 0.992)
and remained nearly unchanged to 97% by day 1 (P = 0.641)
and to 93% by day 3 (P = 0.991). Subsequently, the desmin
content rapidly decreased to 66% in 7-day samples (P = 0.022),
but further significant reduction was not found in 14-day samples
(47%, P = 0.285) (Fig. 5c). Collectively, these results indicated that
desmin degraded more rapidly in PD than in MD samples. Previous
studies proposed that calpain activation and autolysis may
explain the variation of desmin degradation.24 The present results
showed that more rapid -calpain activation and autolysis was
accompanied by more extensive desmin degradation. Therefore,
faster calpain activation and autolysis might cause more rapid
desmin degradation found in PD samples.
In summary, the results showed that the postmortem pH at
the times measured and /m-calpain activity were not different
between PD and MD samples. However, the decrease in -calpain
activity and desmin content was more rapid in PD than in
MD samples during postmortem storage. Therefore, the results
suggested that the postmortem calpain and desmin degradation
were more extensive in PD than in MD breast muscle stored at 5 C.
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