PROCESSING, PRODUCTS, AND FOOD SAFETY
The effect of treating method of pithed pheasant on the content of biogenic
amines in the meat during the course of storage
Z. Hutarova,*1 V. Vecerek,* I. Steinhauserova,† P. Marsalek,* G. Borilova,‡ and P. Forejtek*
*Department of Veterinary Public Health and Toxicology, Faculty of Veterinary Hygiene and Ecology;
†CEITEC—Central European Institute of Technology, and ‡Department of Meat Hygiene and Technology,
Faculty of Veterinary Hygiene and Ecology, University of Veterinary
and Pharmaceutical Sciences, 612 42, Brno, Czech Republic
ABSTRACT The study monitored the effect of various
methods of treating pheasant carcasses after killing on
the hygienic quality of the venison. Pithed pheasants
treated by evisceration (n = 60), drawing (n = 60), or
left untreated (n = 60) were stored for a period of 21
d at temperatures of 0, 7, and 15°C. For determination of biogenic amines, samples of breast and thigh
muscles were taken on d 1, 7, 14, and 21 after killing
of the pheasants. Biogenic amines were separated by
reverse-phase liquid chromatography and consequently
detected by tandem mass spectrometry. The sum of
determined biogenic amine concentrations (cadaverine,
putrescine, histamine, tyramine, tryptamine, phenylethylamine) was compared with the value of the index for meat of high hygienic quality (5 mg/kg). At a
storage temperature of 0°C, the sum of biogenic amine
concentrations did not exceed the value of 5 mg/kg in
either breast or thigh muscle at any time during the
storage period in untreated and drawn pheasants, and
for a period of 14 d in eviscerated pheasants. At a storage temperature of 7°C, values lower than the limit of 5
mg/kg were recorded throughout the storage period in
untreated pheasants, for a period of 14 d of storage in
drawn pheasants, and for a period of just 7 d of storage
in eviscerated birds. At the highest storage temperature (15°C), a value of 5 mg/kg was exceeded in eviscerated and untreated pheasants during the course of the
first week of storage, and in drawn pheasants after the
first week of storage. Our results indicate that the most
suitable method of treatment to ensure high hygienic
quality of the meat (assessed according to concentration of biogenic amines) for the longest period during
the storage of pithed pheasants is to leave the pheasant
carcasses untreated, followed by the drawing, with the
least suitable method being the widely recommended
method of evisceration.
Key words: biogenic amine, hygienic quality, storage temperature, treatment method, pheasant
2013 Poultry Science 92:2182–2187
http://dx.doi.org/10.3382/ps.2012-02906
INTRODUCTION
Pheasant farms are currently operated both for the
purpose of supplying birds for hunting and for slaughtering and subsequent production of meat (Golze, 2010;
Kuzniacka and Adamski, 2010; Kokoszyński et al.,
2012). One way of killing pheasants is to cut the spinal
cord (“pithing”). After killing, the birds’ carcasses may
be treated in 3 various ways. The first method is evisceration (removal of digestive tract through an opening
created by a short cut leading from cloaca in the direction of the sternum; Winkelmayer et al., 2004). The
principal advantage of this method of treatment is the
fact that the digestive tract may be removed complete©2013 Poultry Science Association Inc.
Received November 13, 2012.
Accepted May 10, 2013.
1 Corresponding author: [email protected]
ly without violating the integrity of the guts, thereby
reducing the risk of contamination of the body cavity
by microorganisms penetrating through the wall of digestive tract during the course of storage of the venison. The disadvantage, however, is that evisceration
performed a short time after the bird is killed may lead
to greater bacterial contamination of the surfaces of
the muscles (El-Ghareeb et al., 2009). Another possible
way of treating feathered game after killing is “drawing”
(removing the coils of the intestines through the cloacal opening without violating the integrity of the body
cavity). This is a traditional method used by hunters,
during which the coils of the intestines are wound on a
special hook introduced into the body cavity through
the cloacal opening and subsequently pulled out of the
body. However, this method is considered unsuitable
in terms of hygiene because of the possible tearing of
intestinal coils and the resulting release of digestive
tract contents into the body cavity (Winkelmayer et
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PHEASANT QUALITY RELATED TO BIOGENIC AMINE CONTENT
al., 2004). The final option is to leave the carcasses
of feathered game untreated after killing (the digestive
tract remains in the body cavity; Lecocq, 1997; Paulsen
et al., 2008; Standarova et al., 2012). The advantage
of this method of treatment is that the integrity of the
pheasant carcass is not violated and the digestive tract
is not torn within the body cavity. However, in view
of the possible multiplication and subsequent penetration of enteric bacteria into the body cavity during the
course of storage, this method is also not considered
suitable from the viewpoint of hygiene (Standarova et
al., 2012). The correct and proper treatment of game
after killing has a significant influence on the hygienic
quality of meat.
One indicator of the hygienic quality of meat is the
content of biogenic amines in meat, whose growth is
associated with the multiplication of contaminating
microflora and, thereby, the reduced quality of meat
(Hernandez-Jover et al., 1997; Silva and Gloria, 2002;
Balamatsia et al., 2006; Paulsen et al., 2008; Naila et
al., 2010; Boka et al., 2012). In their work considering
the occurrence of biogenic amine and polyamines in
meat and meat products, Hernandez-Jover et al. (1997)
suggested that the sum of certain biogenic amines
concentrations (cadaverine, putrescine, histamine, tyramine) may be used for assessment of meat hygienic
quality. They determined a value below 5 mg/kg as the
critical concentration for meat of high hygienic quality.
In view of increasing interest in meat quality and
safety from consumers (Nuernberg et al., 2011), studies
focused on the issue of ensuring the hygienic quality
of this product are becoming increasingly important.
The aim of this study was to assess the effect of various treating methods (leaving untreated, evisceration,
drawing), the temperature and storage period of common pheasant carcasses killed by pithing on the hygienic quality of their meat, determined on the basis
of biogenic amine contents (the sum of concentrations
of cadaverine, putrescine, tyramine, histamine, phenylethylamine, and tryptamine). The overall concentrations of polyamines including spermine and spermidine
were not used for the assessment, because they are not
considered as meat quality indicators (Balamatsia et
al., 2006).
MATERIALS AND METHODS
Birds and Their Treatment
Common pheasants (n = 180, male, at the age of 1
yr), slaughtered on the farm, were used to monitor the
effect of treating method on the content of biogenic
amines in meat (breast and thigh muscles) during the
course of storage. The pheasants were killed by pithing
(breaking the spinal cord and subsequent destruction
of the brain).
The carcasses of pithed pheasants were divided into
3 groups of 60 individuals in each according to the
method of treatment. The carcasses in the first group
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were treated in traditional way used by hunters, with
drawing the digestive tract (i.e., by catching the digestive tract in the body cavity and pulling it out using a
special hook introduced into the body cavity through
the cloacal opening). The recommended method for
treatment of feathered game carcasses (i.e., making a
cut in the body cavity and extracting the organs) was
used in the second group. The pheasant carcasses in the
third group were left intact without further treatment
(a common method of treating pheasants in hunting
practice). Each group was further divided into 3 subgroups of 20 individual pheasants in each according to
storage temperature. The first subgroup of pheasants
was placed in a refrigerator commonly used to chill venison set to a temperature of 0°C. The second subgroup
was stored at a temperature of 7°C, and the third one
at a temperature of 15°C.
Samples of breast and thigh muscles were taken from
5 pheasants from each subgroup on d 1, 7, 14, and 21
of storage for determination of biogenic amine concentrations.
In each monitored group and subgroup of pheasant
carcasses, average concentrations of individual biogenic amines determined in the breast and thigh muscles
were added together, and the resulting concentration
compared with the value of 5 mg/kg was considered as
the critical concentration of biogenic amines for meat
of high hygienic quality.
Biogenic Amine Assessment
Measurement of 8 underivatized biogenic amines was
based on high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry.
The modified method of Sagratini et al. (2012) was
used for the determination. Homogenized tissue (0.5 g)
was weighed in a 10-mL glass tube. The samples were
extracted for 1 min with 4 mL of trichloracetic acid
solution (5%) in water with an IKA Genius 3 mixer
(IKA GmbH, Königswinter, Germany) and then in ultrasonic bath (Bandelin Electronic GmbH & Co., Berlin, Germany) for 20 min. The samples were centrifuged
at 800 × g for 10 min at 20°C. Supernatant was filtered
through 0.45-µm nylon filter (Millipore, Billerica, MA)
and used for liquid chromatography-electrospray ionization tandem mass spectrometry analysis. A Thermo
Scientific Hypersil C18 (2.1 mm × 50 mm, 1.9 μm)
column was used at a constant flow rate of 300 μL/
min. The mobile phase consisted of water containing
0.5% formic acid (vol/vol; solvent A) and acetonitrile
containing 0.5% formic acid (solvent B). The gradient
used was a 0 to 2 min linear gradient from 10 to 30% B,
2 to 2.5 min held at 30% B, 2.5 to 3 min from 30 to 10%
B, and 3 to 3.1 min held at 10% B in order for the column to reequilibrate before the next injection. The full
loop injection volume of the tissue extract was set at 10
μL. A Thermo Scientific UHPLC Accela 1250 system
was connected to a Thermo Scientific TSQ Quantum
Access MAX Triple Quadrupole Instrument (Thermo
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Hutarova et al.
Figure 1. Storage at 0°C: the overall concentrations of biogenic amines in the breast and thigh muscles of pheasants treated by evisceration,
drawing, or without treatment. **Statistically highly significant increase (P < 0.01).
Scientific, San Jose, CA) equipped with heated electrospray ionization (HESI-II) probe. The heated electrospray ionization was operated in the positive-ion mode
under the following conditions: capillary temperature,
325.0°C; vaporizer temperature, 300.0°C; sheath gas
pressure, 35.0 psi (241.3 kPa); auxiliary (drying) gas,
10 arbitrary units (arbitrary unit is a relative unit to
show the ratio of amount of gas used by manufacturer/
instrument); and spray voltage, 3,300 V. The optimal
parent/product ion transitions and detection limits of
biogenic amines are shown in Table 1. Standards of
cadaverine, histamine, phenyethylamine, putrescine,
tryptamine, and tyramine as well as trichloroacetic acid
were purchased from Sigma-Aldrich (St. Louis, MO).
All solvents were of residual analysis purity (Chromservis, S.R.O., Praha, Czech Republic).
Statistical Analysis
Results were analyzed using the statistical package
Unistat 5.6. (Unistat Ltd., London, UK). For all variables tested, normality was checked using a ShapiroWilk test (Zar, 1999).
Data, because of their heterogeneous variances, were
subjected to a Kruskal-Wallis ANOVA and subsequently to nonparametric Tukey-type multiple comparison
tests with ranked sums to assess the differences between
all possible pairs of groups (Zar, 1999). A P-value less
than 0.05 was considered as significant, and a P-value
less than 0.01 was considered as highly significant.
RESULTS
The overall concentrations of biogenic amines in the
breast and thigh muscles of the common pheasants
stored at a temperature of 0°C for the individual methods of treating are shown in Figure 1.
It is clear from Figure 1 that at a temperature of
0°C, the concentrations of biogenic amines in the breast
and thigh muscles of the common pheasant carcasses
left without treatment after hunting and those treated
by drawing did not exceed the value considered as the
critical concentration for meat of high hygienic quality
(5 mg/kg) at any time during the storage period (21 d).
In the carcasses of pheasants treated by evisceration,
the limit value of 5 mg/kg was exceeded after d 14 of
Table 1. Optimized parent/product ion transitions and detection limits of the method1
Biogenic amine
Putrescine
Cadaverine
Histamine
Spermidine
Spermine
Tyramine
Phenylethylamine
Tryptamine
Parent ion2
(m/z)
Product ion2
(m/z)
Detection limit
(µg/kg)
89
103
112
146
203
138
122
161
72
86
95
112
112
121
105
144
18
52
27
0.71
0.67
35
3.4
6.2
1Detection limits were calculated as the concentration of the analyte that gives a signal equal to average background plus 3 times the SD of the blank.
2m/z = the mass-to-charge ratio.
PHEASANT QUALITY RELATED TO BIOGENIC AMINE CONTENT
2185
Figure 2. Storage at 7°C: the overall concentrations of biogenic amines in the breast and thigh muscles of pheasants treated by evisceration,
drawing, or without treatment. *Statistically significant increase (P < 0.05). **Statistically highly significant increase (P < 0.01).
storage in thigh muscle. This increase in concentration
of biogenic amines (mainly cadaverine and tyramine)
was statistically significant.
When comparing breast and thigh muscles, the higher values of biogenic amines were found in thigh muscle
in all groups of pheasant carcasses. This difference was
statistically significant (P < 0.01) for values exceeding
the limit of 5 mg/kg (eviscerated pheasants, after 21 d
of storage).
A comparison of the overall concentrations of biogenic amines in the breast and thigh muscles of common
pheasants treated in the 3 different ways and stored at
a temperature of 7°C is shown in Figure 2.
Differences in intensity and speed of increasing in
concentrations of biogenic amines are evident in all
pheasants carcasses stored at a temperature of 7°C.
The overall concentrations of biogenic amines did not
exceed a value of 5 mg/kg at any time during the storage period (21 d) in the case of pheasant carcasses left
untreated following killing. The limit value of 5 mg/kg
was exceeded after 14 d of storage in the thigh muscle
of pheasants treated by drawing (the main increase
was observed in cadaverine and tyramine concentrations). This increase in overall concentrations of biogenic amines was statistically significant (P < 0.05). In
the case of pheasants treated by evisceration, the limit
value was exceeded in the thigh muscle after 7 d of
storage (the main increase was observed in cadaverine
and tyramine concentrations). The increase in overall
concentrations of biogenic amines was statistically significant (P < 0.01).
When comparing breast and thigh muscles, the higher values of biogenic amines were detected in thigh
muscle for all methods of treating pheasants, and this
was statistically different for values exceeding the limit
of 5 mg/kg in eviscerated pheasants on the third date
of sampling (P < 0.01) and in drawn pheasants on the
fourth date of sampling (P < 0.05).
The speed and degree of biogenic amines formation
in the all groups of common pheasants stored at a temperature of 15°C are shown in Figure 3.
A considerable increase in the overall concentrations of biogenic amines is evident in all the monitored
groups at a storage temperature of 15°C. In the case of
pheasant carcasses left without removal of the digestive
tract from the body cavity, the overall concentrations
of biogenic amines exceeded the limit value after 7 d of
storage in both breast and thigh muscles. The increase
in these concentrations was significant (P < 0.01) in
both breast and thigh muscles (the main increase was
observed in cadaverine and putrescine concentrations
in thigh muscle and cadaverine and putrescine concentrations in breast muscle). In the group of pheasants
treated by drawing, the limit value was exceeded by d
7 of storage in both breast and thigh muscles. Increasing these concentrations was significant (P < 0.01) in
both breast and thigh muscles (in both cases the main
increase was observed in cadaverine and tyramine concentrations). In the group of pheasant carcasses treated
by evisceration, the limit value of 5 mg/kg was exceeded by d 7 of storage in thigh muscle. The increase was
statistically significant (P < 0.01; main increase was
observed in cadaverine and tyramine concentrations).
When comparing breast and thigh muscles, the
higher concentrations of biogenic amines were found in
thigh muscle for all methods of treating. A statistically
significant difference was found for values exceeding the
limit of 5 mg/kg in untreated pheasant carcasses on
the third date of sampling (P < 0.05), in eviscerated
pheasants on the second date of sampling (P < 0.01)
and in drawn pheasants on the third date of sampling
(P < 0.05).
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Hutarova et al.
Figure 3. Storage at 15°C: the overall concentrations of biogenic amines in the breast and thigh muscles of pheasants treated by evisceration,
drawing, or without treatment. **Statistically highly significant increase (P < 0.01).
DISCUSSION
The effect of storage temperature and method of
treating pheasants killed by pithing on the increase in
overall concentrations of biogenic amines is clear from
the results obtained by our study. From the viewpoint
of overall concentrations of biogenic amines, the carcasses of common pheasants may be stored at a temperature of 0°C while retaining a high hygienic quality
of meat (when the overall concentrations of biogenic
amines did not exceed the value of 5 mg/kg) for 21 d in
the case of untreated pheasants and pheasants treated
by drawing, and for 14 d in the case of pheasants treated by evisceration.
Pheasant carcasses may be stored at a temperature
of 7°C for a period of 21 d untreated, for 14 d when
treated by drawing, and for 7 d when treated by evisceration.
Pheasant carcasses may be stored at a temperature
of 15°C for a period of 7 d untreated, and just for an
extremely short period of 1 to 2 d when treated by
drawing or evisceration.
The increased concentrations of biogenic amines
found in the meat of pheasants stored at higher temperatures is in accordance with the study previously
described on dependency of storage temperature with
formation of biogenic amines (Shalaby, 1996). Lower
temperatures inhibit the growth of microorganisms and
also reduce their enzymatic activity and, thereby, the
concentration of biogenic amines (Bremer et al., 1998;
Du et al., 2002). When comparing the overall concentrations of biogenic amines in breast and thigh muscles,
we can classify thigh muscle as having a higher risk
from the viewpoint of biogenic amines formation on
the basis of the results obtained. The higher overall
concentrations of biogenic amines in thigh muscle in
comparison with breast muscle were found in all 3 studied groups of pheasants treated in various ways. The
same results were found in meat of wild duck which
their carcasses left intact and treated by drawing after
killing (our unpublished data). Different results were
presented by Standarova et al. (2012), who found the
higher concentrations of biogenic amines in the breast
muscle of shot and pithed pheasants left untreated after
killing. The higher concentrations of biogenic amines
were described in the breast muscle of chickens in comparison with thigh muscle by Silva and Gloria (2002)
and also in breast muscle of wild ducks treated by evisceration, stored at 7 and 15°C (our unpublished data).
The generally recommended method for treating
pheasants is evisceration (Winkelmayer et al., 2004).
Our results indicate, however, that from the viewpoint of the speed and intensity of formation of biogenic amines, the best procedure during the storage of
slaughtered pheasants is to leave the pheasant carcasses
untreated. The second best procedure is the drawing
method traditionally used by hunters, while the least
suitable is the generally recommended method of evisceration. The reason for this may be the higher contamination of the surfaces of muscles occurring during
evisceration which is done in a short period of time
after killing (El-Ghareeb et al., 2009). The evisceration
of pheasants after killing may minimize the risk of penetration and multiplication of microorganisms of the
digestive system, though on the other hand the opening
of the body cavity associated with this method of treatment may represent a pathway for secondary contamination. When unplucked pheasants are stored for a certain time, this pathway for contamination of the muscle
tissue may be highly significant, and according to the
results of this study, the content of biogenic amines in
pheasant breast and thigh muscles represent a greater
PHEASANT QUALITY RELATED TO BIOGENIC AMINE CONTENT
risk than possible contamination by microorganisms
of the digestive tract during treatment by drawing or
leaving the carcasses of pheasants untreated.
ACKNOWLEDGMENTS
This work was supported by the project CEITEC–Central European Institute of Technology
(CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund and the project IGA 93/2011/
FVHE.
REFERENCES
Balamatsia, C. C., E. K. Paleologos, M. G. Kontominas, and I. N.
Sayvaidis. 2006. Correlation between microbial flora, sensory
changes and biogenic amines formation in fresh chicken meat
stored aerobically or under modified atmosphere packing at 4°C:
Possible role of biogenic amines as spoilage indicators. Antonie
Van Leeuwenhoek 89:9–17.
Boka, B., N. Adanyi, D. Virag, M. Sebela, and A. Kiss. 2012. Spoilage detection with biogenic amine biosensor of different enzyme
electrodes. Electroanalysis 24:181–186.
Bremer, P. J., C. M. Osborne, R. A. Kemp, P. V. Veghel, and G.
C. Fletcher. 1998. Thermal death times of Hafnia alvei cells in
a model suspension and in artificially contaminated hot-smoked
kahawai (Arripis trutta). J. Food Prot. 61:1047–1051.
Du, W. X., C. M. Lin, A. T. Phu, J. A. Cornell, M. R. Marshall,
and C. I. Wei. 2002. Development of biogenic amines in yellowfin
tuna (Thunnus albacares): Effect of storage and correlation with
decarboxylase-positive bacterial flora. J. Food Sci. 67:292–301.
El-Ghareeb, W. R., F. J. R. Smulders, A. M. A. Morshdy, R. Winkelmayer, and P. Paulsen. 2009. Microbiological condition and
shelf life of meat from hunted game birds. Eur. J. Wildl. Res.
55:317–323.
Golze, M. 2010. Fasanenproduktion zur Fleischgewinnung
und zum Auswildern. Rundschau fűr Fleischhygiene and
Lebensmittelűberwachung. 62:9–12.
Hernandez-Jover, T., M. Izquierdo-Pulido, M. T. Veciana-Nogues,
A. Marine-Font, and M. C. Vidal-Carou. 1997. Biogenic amine
2187
and polyamine contents in meat and meat products. J. Agric.
Food Chem. 45:2098–2102.
Kokoszyński, D., Z. Bernacki, and L. Duszyński. 2012. Body conformation, carcass composition and physicochemical and sensory
properties of meat from pheasants of different origin. Czech J.
Anim. Sci. 57:115–124.
Kuzniacka, J., and M. Adamski. 2010. Growth rate of body weight
and measurements in pheasants reared up to the 24th week of
life. Arch. Tierz. 53:360–367.
Lecocq, Y. 1997. A European perspective on wild game meat and
public health. Rev. Sci. Tech. 16:579–585.
Naila, A., S. Flint, G. Fletcher, P. Bremer, and G. Meerdink. 2010.
Control of biogenic amines in food-existing and emerging approaches. J. Food Sci. 75:139–150.
Nuernberg, K., J. Slamecka, J. Mojto, J. Gasparik, and G. Nuernberg. 2011. Muscle fat composition of pheasants (Phasianus colchicus), wild ducks (Anas platyrhynchos) and black coots (Fulica
atra). Eur. J. Wildl. Res. 57:795–803.
Paulsen, P., J. Nagy, P. Popelka, V. Ledecky, S. Marcincak, M. Pipova, F. J. M. Smudlers, P. Hofbauer, P. Lazar, and Z. Dicakova.
2008. Influence of storage conditions and shotshell wounding on
the hygienic condition of hunted, uneviscerated pheasant (Phasianus colchicus). Poult. Sci. 87:191–195.
Sagratini, G., M. Fernandez-Franzon, F. De Berardinis, G. Font, S.
Vittori, and J. Manes. 2012. Simultaneous determination of eight
underivatised biogenic amines in fish by solid phase extraction
and liquid chromatography-tandem mass spectrometry. Food
Chem. 132:537–543.
Shalaby, A. R. 1996. Significance of biogenic amines to food safety
and human health. Food Res. Int. 29:675–690.
Silva, C. M. G., and B. A. Gloria. 2002. Bioactive amines in chicken
breast and thigh after slaughter and during storage at 4±1°C and
chicken-based meat products. Food Chem. 78:241–248.
Standarova, E., L. Vorlova, and L. Gallas. 2012. Distribution of
biogenic amines and polyamines in the pheasant meat. Maso.
1:51–54.
Winkelmayer, R., P. Lebersorger, and H. F. Zedka. 2004. Aufbrechen—Ausweiden. Pages 69–88 in Wildbret-Hygiene: Das Buch zur
Wildfleisch-Verordnung. Zentralle Osterr. Landesjagdverbände,
Wien.
Zar, J. H. 1999. Biostatistical Analysis. 4th ed. Prentice Hall, Upper
Saddle River, NJ.