The effect of transportation on the expression of heat shock proteins
and meat quality of M. longissimus dorsi in pigs
Jimian Yu, Shu Tang, Endong Bao*, Miao Zhang, Qingqing Hao, Zhenghua Yue
College of Veterinary Medicine, Nanjing Agricultural University, Weigang 1, Nanjing 210095, China
a r t i c l ei n f o
Received 5 November 2008
Received in revised form 15 June 2009
Accepted 17 June 2009
Heat shock proteins
a b s t r a c t
This study investigates the effect of different transport times on meat quality and the correlation between
meat quality and Hsp expression in M. longissimus dorsi (LD) of pigs. After transportation for 1 h, 2 h or
4 h, respectively, blood plasma creatine kinase (CK) and lactate dehydrogenase (LDH) increased. The
LD meat from 1 h and 2 h transported pigs had lower initial and ultimate pH values (pHi and pHu, respec-
tively), higher drip loss and L*values compared to controls, indicating a higher likelihood of pale, soft and
exudative (PSE) meat. Meat quality was lower after 2 h compared to 1 h or 4 h of transport. All four Hsps
tested (alpha-B-crystalline, Hsp27, Hsp70 and Hsp90) by ELISA in the LD tissue of pigs tended to decrease
after transportation. One possible mechanism resulting in poor meat quality in the LD after transport
seems to be a decline in Hsp expression.
? 2009 Elsevier Ltd. All rights reserved.
Research has shown that transport duration is an important fac-
tor affecting animal welfare and meat quality in pigs (Lammens
et al., 2007; Pérez et al., 2002; Shen et al., 2006; Warriss, Brown,
Edwards, & Knowles, 1998). The mortality of pigs transported to
slaughter increases as the transportation distance increases (Gosá-
lvez, Averós, Valdelvira, & Herranz, 2006). Numerous factors can
influence meat quality, including weather conditions, loading and
unloading, and food and water deprivation (Abbott et al., 1995;
dalla Costa et al., 2007; Mota-Rojas et al., 2006; Nanni Costa, Lo
Fiego, Dall’Olio, Davoli & Russo, 1999). Guàrdia et al. (2004) dem-
onstrated that the risk of pale, soft, exudative (PSE) meat in pigs in-
creases with stocking density during transport for transits longer
than 3 h, while the opposite occurs for shorter transits. Subse-
quently, Guàrdia et al. (2005) found that the risk of dark, firm
and dry (DFD) meat increases with high stocking density and lai-
rage time, and with on-farm fasting times longer than 22 h. It
was reported that damage in the muscle membrane is one of the
primary reasons for deterioration in meat quality (Cheah, Cheah,
& Krausgrill, 1995; Gray, Gomaa, & Buckley, 1996; Pliquett, Alt-
mann, Pliquett, & Schöberlein, 2003). Research has also shown that
heat shock proteins (Hsps) are a group of evolutionarily conserved
proteins that are synthesized in response to physical, chemical or
biological stresses, including transport stress (Bao, Sultan, Nowak,
& Hartung, 2008; Benjamin & McMillan, 1998; Li, Bao, Wang, &
Zhao, 2007; Yu, Bao, Zhao, & Lv, 2007). Hsps play an important role
as molecular chaperones during protein assembly (Haslbeck,
Miess, Stromer, Walter, & Buchner, 2005), protein folding and
unfolding (Zietkiewicz, Krzewska, & Liberek, 2004), and in the
refolding of damaged proteins (Marques et al., 2006) for the pro-
tection and repair of cells and tissues. A variety of stresses, includ-
ing oxidative stress, bacterial infection, calorie restriction, and
resistance training have been shown to increase the expression
of Hsps in skeletal muscle (Escobedo, Pucci, & Koh, 2004; Murlasits
et al., 2006; Ramaglia, Harapa, White, & Buck, 2004; Selsby, Judge,
Yimlamai, Leeuwenburgh, & Dodd, 2005). Expression of Hsp70 was
no different in skeletal muscle of homozygous recessive (nn) pigs
compared to homozygous dominant (NN) or to heterozygous
(Nn) animals, although homozygous recessive pigs manifested
many of the traditional stress-induced meat quality defects (van
Laack, Faustman, & Sebranek, 1993). However, Bao, Sultan, Nowak,
and Hartung (2001) concluded the expression of Hsp90 markedly
decreased in M. longissimus dorsi (LD) of pigs following 6 h of
In China, the Erhualian pig is an eminent regional breed. It is
famous not only for its high breeding capability, but also for strong
stress resistance. Crossbreeding between the Pietrain and Erhua-
lian breeds has been used to obtain animals with high lean muscle
ratio and strong stress resistance. It is unknown whether the
expression of Hsps, and other factors effecting meat quality, are
different in the LD of F2 offspring of Pietrain and Erhualian parents
following different times of transportation. The purpose of this
study is to evaluate the effect of different transportation times
on meat quality and Hsp level, and to correlate meat quality with
Hsp expression in the LD of transported F2 offspring of Pietrain
and Erhualian parents.
0309-1740/$ - see front matter ? 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +86 2584395316; fax: +86 2584398669.
E-mail address: firstname.lastname@example.org (E. Bao).
Meat Science 83 (2009) 474–478
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/meatsci
2. Materials and methods
2.1. Animals, experimental design, and sampling
Twenty experimental pigs were the castrated F2 offspring of
Pietrain and Erhualian parents. The animals were reared in four
individual pens (2.5 ? 4.0 m2, 5 pigs per pen) at Jiangsu Academy
of Agricultural Sciences (Nanjing city, China) and given access to
a commercial pig feed and water ad libitum. All pigs were raised
up to a live weight of 50 ± 1 kg by the same person and were ran-
domly divided into one control group and three transport groups
(5 pigs per group). Each group was housed in a separate pen. On
the day of the transport trial, the experimental pigs were loaded
onto a truck and taken on a continuous journey for 1 h, 2 h or
4 h at 30–40 km/h, while the control group was kept under normal
conditions. The transport route included town traffic and state
roads. Water was continually available during transportation. At
the end of the journey, the pigs were stunned electrically using
operator-handled tongs (100 V, 5 s) and all the specimen were col-
lected within 20 min of arrival. Immediately after being stunned,
all pigs were sacrificed by exsanguination from the carotid artery
and jugular vein. Blood samples were collected from the carotid ar-
tery and jugular vein during exsanguination. Blood samples
(10 mL) were transferred into collection tubes containing heparin
anti-coagulant (50 IU mL?1), and immediately chilled on ice. Plas-
ma samples for subsequent enzyme determination were obtained
by centrifugation of whole blood at 1500 rpm for 5 min and were
frozen and stored at ?80 ?C. For quantitation of the Hsps, a speci-
men of each LD muscle sample (0 min) was taken as soon as pos-
sible after sticking from the right part of LD between 8th and
13th thoracic vertebra (sample 0 min) and quickly placed into
1.5 mL tubes, frozen in liquid nitrogen, and then stored at
?80 ?C. Further muscle samples (0 min) were immediately col-
lected and stored at 4 ?C for subsequent meat quality analysis. Also,
1–2 g of muscle was collected at 45 min and 24 h post mortem and
stored at ?80 ?C for the measurement of pH immediately (pHi) and
pH ultimately (pHu).
The study followed the guidelines of the regional Animal Ethics
Committee and was approved by the Institutional Animal Care and
Use Committee of Nanjing Agricultural University.
2.2. Measurement of some blood parameters
The activities of plasma creatine kinase (CK) and lactate dehy-
drogenase (LDH) were assessed using a commercial kit modified
for use with a multiwell plate spectrophotometer as previously de-
scribed by Sun, Liu, Zhao, Bao, and Wang (2007).
2.3. Measurement of some meat quality parameters
2.3.1. pH value
One gram of LD specimen, sampled at 45 min and 24 h post-
mortem, was homogenized in 9 mL of 5 mM iodoacetate solution
and the pH of the homogenate was measured using a glass elec-
trode connected to a pH meter (Fernandez, Neyraud, Astruc, & San-
te, 2002). The pH meter was standardized by a two-point method
against buffer standards of pH 6.86 and pH 9.18.
2.3.2. Drip loss
A cored LD sample, 4 cm in diameter, was collected and
weighed. Subsequently, the sample was suspended from a steel
wire hook within a polyethylene plastic bag. Samples were stored
at 4 ?C for 24 h. After 24 h, samples were reweighed, and drip loss
was calculated as (final weight of sample-initial weight of sample)/
initial weight of sample ? 100.
2.3.3. Expressible moisture
A cored LD sample, 2 cm in diameter and 0.5 cm in thickness,
was collected and weighed. Subsequently, the core was pressed
by a force meter at 35 kg force for 5 min. Samples were reweighed,
and expressible moisture was calculated as (final weight of sam-
ple-initial weight of sample)/initial weight of sample ? 100.
2.3.4. Cooking loss and shear value
While still frozen, each LD fillet was immersed, in cooking bags,
in a boiling water bath until reaching an internal endpoint of 70 ?C
(approximately 20 min). The temperature was measured with
cooking thermometer inserted into the thickest part of the fillet.
After cooking, the samples were cooled in an ice bath for 30 min
and were reweighed, and the cook loss was calculated as (final
weight of fillet-initial weight of fillet)/initial weight of fillet ? 100.
The cooked fillets (1 ? 1 ? 1 cm3) were then used for shear mea-
surements. Shear values were determined using the Warner–Brat-
zler shearing device.
2.3.5. Meat color
Color (L*= lightness, a*= redness, b*= yellowness) was mea-
sured with a Minolta surface spectrocolorimeter (model CR200,
illuminant D65 and viewing diameter 8 mm). LD muscles were ex-
posed to air for at least 10 min at 15 ?C before color measurement.
Color, L*, a*, and b*, values were recorded and averaged at three
locations of the thickest part of the ventral surface in the middle
of the LD muscles.
2.4. Semiquantitative detection of Hsps by enzyme-linked
immunosorbent assay (ELISA)
After complete washing in ice-cold physiological saline, the
muscle samples were homogenized on ice in 10 volumes of
homogenization buffer [0.15 M NaCl, 20 mM Tris–HCl (pH 8.0),
1 mM EDTA, 1 mM PMSF, 0.1 lM E-46, 0.08 lM aprotinin, 0.1 lM
leupeptin, and 0.1% NP-40] (Shaila et al., 2005) using an Ultra-Tur-
rax homogenizer, and the homogenates were centrifuged at
12,000 rpm for 20 min at 4 ?C to remove cellular debris. The super-
The levels of alpha-B-crystalline, Hsp27, Hsp70 and Hsp90
in theLDmuscles ofboth
were measured using commercially available ELISA kits (QRCT-
line; QRCT-30123011EIA/UTL, goat anti-porcine Hsp27; QRCT-
32232101EIA/UTL, goat anti-porcine Hsp70; QRCT-3222021EIA/
UTL, goat anti-porcine Hsp90, Adlitteram Diagnostic Laboratories,
USA.). Quantification of samples was performed using a standard
curve. b-actin (QRCT-3222211EIAUTL, goat anti-porcine b-actin,
Adlitteram Diagnostic Laboratories, USA) was used to control for
the bias caused by the procedure of protein extraction. Immuno-
logical detection of the proteins was performed essentially
according to the manufacturer’s instructions. The quantity of
Hsp in each sample was normalized using the following formula:
transported andcontrol pigs
Relative quantity of Hsp ¼ quantity of Hsp=quantity of b-actin
2.5. Statistical analysis
Statistical analysis of the differences between each group was
carried out using a one-way analysis of variance (ANOVA) using
the Statistical Package for Social Sciences (SPSS version 11.5). Com-
parison of the mean value of the control group with others was
performed by the Least Significant Difference (LSD) test. Differ-
ences were regarded as significant at P < 0.05.
J. Yu et al./Meat Science 83 (2009) 474–478
3.1. Concentrations of some blood parameters
The concentrations of CK and LDH in the blood pasma which are
associated with cellular injuries are shown in Fig. 1. The activities
of plasma CK and LDH gradually increased shortly after the pigs
were exposed to transportation of 1 h and they were maintained
at a high level until the termination of transportation. The plasma
CK activities exhibited a sharp induction after 1 h (P < 0.01), which
was maintained at 2 h (P < 0.01) of transportation. However, there
was no statistical increase in plasma CK after 4 h of transportation
compared to the control. The concentration of plasma LDH showed
a significant induction after 1 h (P < 0.01) of transportation. How-
ever, there was no statistical increase after 2 h or 4 h in the trans-
port groups compared to the control group.
3.2. Meat quality parameters
The quality characteristics for the LD meat of the pigs are pre-
sented in Table 1. After 1 h of transportation, the pHi value
(P < 0.01) and pHu value (P < 0.05) in the LD of the transported pigs
decreased compared to control pigs. Furthermore, both the pHi and
pHu values decreased significantly (P < 0.01) after 2 h of transpor-
tation, where they reached their minimum levels. However, no sta-
tistical reduction of pHi or pHu was found in the LD of pigs after 4 h
of transportation compared to the control pigs. The drip loss of the
pig LD decreased greatly in the transported lines. Compared with
control pigs, the drip loss (P < 0.01) and expressible moisture
(P < 0.05) of the LD in the transported groups were significantly
higher after 1 h of transportation and highest after 2 h (P < 0.01)
of transportation. After 4 h of transport, there was no statistically
significant difference in drip loss or expressible moisture between
the transported pigs and the controls. A statistically significant ele-
vation of cooking loss was demonstrated in transported pigs after
1 h (P < 0.01) when compared to the controls. The shear values of
LD decreased slightly in the transported pigs after 1 h of transpor-
tation. A significant reduction of the shear value in the pig LD was
demonstrated after 2 h (P < 0.01) of transportation. No statistical
difference was found between the shear values of pigs transported
for 4 h and the control group. The meat color L*value and b*value
increased slightly with transportation. A significant induction of L*
value was demonstrated at both 1 h (P < 0.01) and 2 h of transpor-
tation (P < 0.01), and a clear induction of b*value was detected at
2 h of transportation (P < 0.01). However, compared with the con-
trol pigs, the color a*value decreased significantly (P < 0.01) in the
LD of the pigs transported for 4 h.
3.3. The expression levels of the four tested Hsps
The expression data for Alpha-B-crystalline, Hsp27, Hsp70 and
Hsp90 in the LD, normalized to b-actin, are shown in Fig. 2. Gener-
ally, the levels of the four tested Hsps decreased after transporta-
tion in the LD tissue of F2 offspring of Pietrain and Erhualian
parents. The level of alpha-B-crystalline decreased significantly
(P < 0.01) after 1 h, but recovered (P > 0.05) after 2 h of transporta-
tion compared with the controls. After 4 h of transportation, the le-
vel of alpha-B-crystalline was clearly lower (P < 0.01) than the
control. The level of Hsp27 decreased significantly and reached
its minimum level after 1 h of transportation (P < 0.01). After 2 h
and 4 h of transportation, the level of Hsp27 increased slightly
when compared to the level measured after 1 h transportation,
but were still lower at 2 h (P < 0.01) and 4 h (P < 0.05) of transpor-
Fig. 1. Effects of the duration of transport on plasma CK and LDH in pigs.**P < 0.01,
when comparing the transported groups with the control. Values indicated are
mean ± SD.
Effects of the duration of transport on pig M. longissimus dorsi quality.
Transport time (h)
Drip loss (%)
Expressible moisture (%)
Cooking loss (%)
5.830 ± 0.062
5.543 ± 0.092
1.913 ± 0.408
37.268 ± 2.188
18.130 ± 2.055
57.516 ± 6.11
48.726 ± 2.248
10.396 ± 0.855
0.633 ± 0.346
5.604 ± 0.045**
5.418 ± 0.051*
2.672 ± 0.278**
39.599 ± 1.72*
24.615 ± 1.419**
53.900 ± 8.40
52.148 ± 2.320**
9.977 ± 0.928
0.953 ± 0.328
5.188 ± 0.103**
5.088 ± 0.057**
3.040 ± 0.402**
40.715 ± 2.602**
20.05 ± 2.415
48.236 ± 6.66**
52.548 ± 2.150**
10.149 ± 0.516
2.073 ± 0.507**
5.684 ± 0.127
5.472 ± 0.083
1.986 ± 0.359
38.774 ± 2.194
17.964 ± 1.832
60.701 ± 7.80
49.660 ± 2.857
9.708 ± 0.745*
1.018 ± 0.348
aWarner–Bratzler shear force value.
*P < 0.05 when comparing the transported groups with the control.
**P < 0.01 when comparing the transported groups with the control. Values indicated are mean ± SD.
Fig. 2. Effects of the duration of transport on the expression of alpha-B-crystalline,
Hsp27, Hsp70 and Hsp90 in pigs.
transported groups with the control. Values indicated are mean ± SD.
*P < 0.05,
**P < 0.01, when comparing the
J. Yu et al./Meat Science 83 (2009) 474–478
tation than in the non-transported pigs. Hsp70 expression showed
a slight decrease after 1 h and 2 h of transportation when com-
pared to the control. A significant reduction in Hsp70 occurred
after 4 h of transportation (P < 0.05). The amount of Hsp90 de-
creased significantly (P < 0.01) in transported pigs after 1 h of
transportation only. After 2 h and 4 h, the levels of Hsp90 were
at the levels of the controls.
Blood components, such as CK, LDH, corticotrophin, cortisol,
insulin, triiodothyronine (T3) and thyroxine (T4), are often used to
assess stress conditions in animals (Fazio, Medica, Alberghina, Cav-
aleri, & Ferlazzo, 2005; Knowles, 1999; Li et al., 2008; Warriss et al.,
1995; Yu et al., 2007). In the current study, CK activity was found to
clearly increase (P < 0.01) in the plasma of F2 offspring pigs of Pie-
train and Erhualian parents after 1 h or 2 h of transportation. Such
increases indicate muscle damage and result from disruption in
muscle cell membrane (sarcolemma) function and permeability
(Fabrega et al., 2002). However, no statistical increase of plasma
CK was observed in pigs after 4 h of transportation compared to
the controls. This seems to indicate that cellular membrane damage
may be alleviated in muscles of pigs during 4 h of transportation.
The experiments also demonstrate that the activity of plasma LDH
increases significantly after 1 h of transportation. Under anaerobic
conditions, pyroracemic acid was catalyzed by LDH and hydroge-
nized as lactic acid, which is the end product of glycolysis. The en-
zyme LDH plays an important role in glycolysis and the induction
of plasma LDH activity in the present study may indicate the switch
from aerobic to anaerobic glycolysis in the transported pigs.
After 1 h or 2 h of transportation, the LD from F2 offspring of
Pietrain and Erhualian parents show lower pHi and pHu values,
higher drip loss and expressible moisture compared to the control
pigs. Moreover, the L*values and b values from the transported
pigs are much higher (P < 0.01) than those from control pigs, indi-
cating a higher likelihood of PSE meat, which is similar to the re-
sults of previous research (Mota-Rojas et al., 2006; Shen et al.,
2006). Shen et al. (2006) concluded that pre-slaughter transport
accelerates ATP depletion, which leads to lower energy status of
the post mortem muscle immediately post-exsanguination when
compared with control muscles. The lower energy status led to
AMP-activated protein kinase activation within 1 h postmortem,
subsequently increasing glycolysis, and resulting in a high inci-
dence of PSE meat. Recent evidence suggests that degradation of
key cytoskeletal proteins by calpain proteinases plays a role in
determining water-holding capacity (Huff-Lonergan & Lonergan,
2005). Calpains degrade muscle proteins at a faster rate at higher
tissue pH (Huff-Lonergan & Lonergan, 1999; Kendall, Koohmaraie,
Arbona, Williams, & Young, 1993). A low tissue pH would depress
the activity of calpains, therefore, proteins that link myofibrils to-
gether, and to the cell membrane, are not degraded, leading to an
increased drip loss and undesirable water-holding capacity. Addi-
tionally, when the pH value in the meat is greater than the isoelec-
tric point of the myofibrillar proteins, water molecules are tightly
bound, causing more light to be absorbed by the muscle; hence,
the meat appears darker in color (Cornforth, 1994). In our study,
the LD muscle exhibited a lower water-holding capacity and the
higher L*and b values may be the consequence of the reduction
in pH. In transported pigs, the shear force value of the LD decreased
slightly after 1 h of transport, and it was significantly reduced after
2 h (P < 0.01) of transportation. The present study also confirmed
that the levels of Hsp27 and alpha B-crystallin decrease signifi-
cantly after different periods of transportation. Down-regulation
of Hsp27 can actually accelerate actin disorganization or degrada-
tion, a process that is related to the tenderness of the meat (Ber-
nard et al., 2007; Morzel, Terlouw, Chambon, Micol, & Picard,
2008). Hsp27 positively correlates with Warner–Bratzler (WB)
shear force (Kim et al., 2008). Therefore, it appears that the loss
of myofibrillar-related protein function and the subsequent reduc-
tion of myofibril integrity can explain the increase in meat tender-
ness. These results suggest that the decrease of Hsp27 in the LD of
F2 offspring of Pietrain and Erhualian parents may increase the
degradation of muscle proteins such as actin and myosin and, con-
sequently, improve meat tenderness. Meat tenderization was also
reported as the result of ultrastructural changes caused by a weak-
ening of myofibril integrity in the muscle at the junction of the Z-
line and the I-band (Ho, Stromer, & Robson, 1996; Taylor, Geesink,
Thompson, Koohmaraie, & Goll, 1995).
Our results find the greatest decline in pH, drip loss, and
expressible moisture, and the greatest inductions in L*and b val-
ues, in the pigs transported for 2 h, indicating that pigs subjected
to 2 h of transportation show signs of a reduced meat quality com-
pared to pigs subjected to either 1 h or 4 h transportation. The
present study also concludes that the LD quality of the investigated
pigs declines as the transportation time is prolonged. Gispert et al.
(2000) found a lower proportion of acceptable meat quality in pigs
subjected to transports longer than 2 h compared to shorter trans-
ports (<2 h). Pérez et al. (2002) concluded that under normal Span-
ish commercial conditions, pigs subjected to short transport times
of 30 min showed a more intense stress response and poorer meat
quality than pigs subjected to a transport time of 3 h when they
were immediately slaughtered on arrival at the slaughterhouse.
The results of Pérez et al. (2002) suggest that transports longer
than 3 h may allow the animals to adapt and seems to have a sim-
ilar effect as a resting period in the lairage. However, Warriss, Dud-
ley, and Brown (1983) found that transport time did not,
ultimately, have an effect on muscle pH. In an extensive review
of the ultimate causes of pH variation in pigs, Fernandez and Torn-
berg (1991) concluded that it is difficult to draw a conclusion
regarding the effect of transport time because of the different re-
sults obtained in different studies, and that this might point to that
the existence of several interacting factors. Nevertheless, the pres-
ent study found no detrimental effect at 4 h of pre-slaughter trans-
portation on meat quality which seems to confirm the results of
the study by Pérez et al. (2002) who suggested an adaptation of
pigs when transported longer than 3 h.
All four Hsps tested (alpha-B-crystalline, Hsp27, Hsp70 and
Hsp90) by ELISA in the LD tissue of pigs exhibited a decreasing ten-
dency following different times of transportation, similar to the re-
sults of Bao et al. (2001). Because of the marked increases in
plasma CK and LDH activities, which indicate pig muscle damage
and disruptions in muscle cell membrane function and permeabil-
ity in the transported pigs, it is presumed that the decrease in Hsp
expression may be disadvantageous for the recovery of the normal
function of the cell membrane and protection of disrupted cell
homeostasis after different times of transport. Furthermore, the
combination of high temperature at low pH or abnormally low ulti-
mate pH causes denaturation of sarcoplasmic and myofibrillar pro-
teins, resulting in a paler color and reduced drip loss (Scheffler &
Gerrard, 2007). van de Wiel and Zhang (2007) concluded that des-
min is a marker protein for water-holding capacity and desmin lev-
els are higher in pig muscle with low drip loss. Desmin is a
cytoskeletal protein, which is responsible for the integrity and
rigidity of the muscle cell. Therefore, the decrease in Hsps in the
LD of transported pigs, as observed in the present study, may be
disadvantageous for maintaining muscle cell integrity and repair-
ing denatured proteins, such as desmin, and sarcoplasmic and
myofibrillar proteins, which could subsequently lead to reduced
meat color and water-holding capacity. There are many proteins
related to the meat quality of the LD in pigs (Hwang, Park, Kim,
Cho, & Lee, 2005). The current results imply that a possible
J. Yu et al./Meat Science 83 (2009) 474–478
mechanism for producing poor meat quality in the LD after differ- Download full-text
ent times of transportation may be the decline of Hsp expression.
This work was supported by Grants (30430420, 30170682, and
30571400) from the National Science Fund of China.
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