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1
-globulins,
2
-globulins and -globulins (Kohn 1997).
However, in the disease such as in equine babesiosis, the
serum concentration of the proteins can change significantly
(Kumar et al 2002).
Equine babesiosis is a tick-borne protozoal disease caused
by Babesia caballi and Babesia equi. Although it has been
reported that horses are most susceptible to infection, the
disease can affect horses, donkeys, their hybrids and wild
equidae (Okamura et al 2005, 2007). The disease may take
on an acute or chronic form (Radostits et al 1999). Clinical
signs of the acute form include weakness, anorexia, fever
of up to 42 C, which usually becomes irregular after the
second day (Malikides et al 2000). Animals that have the
chronic form of the illness usually survive for months without
any apparent signs of the disease. Symptoms may include
steady weight loss and slight anaemia usually following
exercise or stress situations (Hailat et al 1997).
Babesiosis is an important disease from both economic
and animal health point of view, because it involves
substantial losses by death and by decrease in animal
production. In addition, the equestrian legislation of some
countries do not allow seropositive symptomatic and non-
symptomatic animals (Asenzo et al 2008).
Horses with babesiosis often display an increase in the
concentration of total proteins usually caused by dehydration
and by the increase in the -globulin concentration (Kumar
et al 2002). However, until now zone-electrophoresis
to evaluate changes in protein fractions has not been
applied to the serum in equine babesiosis, although it is
very useful from a clinical point of view because it is less
time consuming.
The aim of the present work was characterize the
serum electrophoretic pattern in horses suffering from
babesiosis and to evaluate its application in the diagnosis
by the study of the quantitative alterations in the serum
proteins associated to this disease.
174
R BARRERA ET AL
MATERIALS AND METHODS
ANIMALS
Fifty three adult horses of different breed, sex, and
age were studied and classified in two groups. Group I
included 19 healthy horses all seronegative to babesiosis.
Animals from Group II were presented to the Veterinary
Teaching Hospital at Extremadura University (Spain)
showing clinical symptoms consistent with the disease
(table 1). This group included 34 horses suffering from
naturally acquired babesiosis: 7 horses (20.59%) showed
positive antibody titre against Babesia caballi, 9 horses
(26.47%) against Babesia equi and 18 horses (52.94%)
showed positive titre against both Babesia species (Table2).
Disease was always diagnosed or ruled out by indirect
immunofluorescence antibody assay.
SEROLOGICAL TESTS
Serological tests were performed according with
Callow et al (1979), Donnelly et al (1980
a, b
), Soule
et al (1984), and Habela et al (1989). Antigen of Babesia
equi was prepared after esplenectomized carrier horse
(titre 1/320) from Andaluca (local strain), and the antigen
of Babesia caballi after infection with USDA strain of
seronegative horse, which was kindly provided for Professor
G. Uilenberg (Veterinary Faculty, Utrecht, Netherlands).
Commercial fluorescein conjugated rabbit anti-horse
immunoglobulin (Nordic, Tilburg, Netherlands) was used
at a dilution of 1:60 in phosphate-buffered saline. A titre
of 1:80 was considered positive. Positive and negative
control sera were kindly supplied by Professor G. Uilenberg
(Veterinary Faculty, Utrecht, Netherlands).
SERUM TOTAL PROTEIN
Blood samples were obtained under non-stress conditions
by jugular venipuncture using vacutainer tubes. Serum was
obtained by centrifugation for 10 minutes at 600 g using
serum-separator tubes. Serum was used instead of plasma in
order to avoid interference from fibrinogen. Serum protein
concentration was calculated by Biurets method (Gornall
et al 1949). Freeze-dried bovine serum albumin (Qumica
Clnica Aplicada, Spain) was used as a control.
SERUM ELECTROPHORESIS
To characterize the serum electrophoretic pattern in
the animals studied, serum electrophoresis was performed.
Zone electrophoresis (Kohn 1957) on a supporting medium
of cellulose acetate strips (2.5 17 cm) (Cellogel
, Atom
Biosystems, Spain) was performed. Serum proteins
migrated in an electric field of 200 volts for 65 minutes.
The buffering solution used was sodium veronal 0.04 M
and sodium-dietilbarbiturate 0.82 g/dl. Amido-Black
Table 1. More significant clinical manifestations noted in the
Group II (horses with babesiosis).
Manifestaciones clnicas ms significativas observadas en el
Grupo II (caballos con babesiosis).
Horse
N
Weakness Anorexia Fever*
Steady
weight
loss
Pale
mucous
mem-
branes
Jaundice Oedemas
1 +
2 + + + +
3 + + + + +
4 + + + +
5 +
6 +
7 + + + + +
8 + + + + +
9 +
10 + +
11 + + + +
12
13 + +
14 +
15 + + + + + + +
16 + + + + +
17 +
18 + + + + +
19 + + +
20 +
21 + + +
22 +
23 + + + + +
24
25 + + + +
26 + + + +
27 + + +
28 + +
29 + + + + +
30 + + + + + +
31 + + + +
32 + + + + +
33 + + + +
34 + + + + +
* Body temperature up to 39 C.
(Atom Biosystems, Spain) staining was used, and a
solution of methanol, acetic acid, and water (40:10:50) was
applied as a destaining solution. Finally, the strips were
analyzed in a scanner densitometer using the Ultroscan
GSX software (Pharmacia LKB Biotechnology, San
Francisco, CA, USA).
STATISTICAL ANALYSIS
Statistical analysis of the results was performed using
the non-parametric of Mann-Whitney U test. The used
software was SPSS 14 (SPSS, Inc., Chicago, IL, USA).
175
ELECTROPHORESIS, SERUM, BABESIOSIS, HORSE
Table 2. Antibody titers for Babesia equi and Babesia caballi obtained by indirect immunofluorescence test (IFI) in the Group II
(horses with babesiosis).
Ttulos de anticuerpos frente a Babesia equi y Babesia caballi obtenidos mediante inmunofluorescencia indirecta (IFI) en el Grupo II (caballos
con babesiosis).
Horse N B. equi B. caballi Horse N B. equi B. caballi
1 1/160 1/160 18 1/1280 Negative
2 1/640 1/320 19 1/160 1/160
3 1/160 1/320 20 1/320 Negative
4 1/80 1/320 21 1/320 Negative
5 1/160 1/160 22 Negative 1/640
6 Negative 1/640 23 1/160 Negative
7 1/320 1/1280 24 1/80 Negative
8 Negative 1/640 25 1/160 1/320
9 Negative 1/160 26 1/160 Negative
10 1/640 1/160 27 1/80 1/640
11 Negative 1/320 28 1/640 Negative
12 1/80 1/80 29 1/1280 1/640
13 1/640 Negative 30 1/80 1/640
14 Negative 1/320 31 1/160 1/320
15 1/320 1/320 32 1/1280 1/160
16 Negative 1/1280 33 1/160 1/80
17 1/160 Negative 34 1/320 1/160
RESULTS
GROUP I
Serum electrophoresis from Group I showed a normal
electrophoretic pattern, characterized by six different
bands: albumin,
1
-globulin,
2
-globulin,
1
-globulin,
2
-globulin and -globulin fractions (figure 1). Statistical
analysis results of serum protein fractions from Group I
are shown in table 3.
GROUP II
In our study, the median obtained for the serum total
proteins in the Group II was 7.18 g/L (interquartile range
(IQR) 5.80-9.03 g/L), showing a statistically significant
difference (P < 0,001) with the Group I (table 3). Although,
the range of values obtained was very wide (minimum
value = 36g/L; maximum value = 142.7 g/L), hyperproteinemia
was observed in 14 horses. Conversely, six animals showed
hypoproteinemia with a serum total protein concentration
lower than 55 g/L.
Although the median albumin concentration was
3.96 g/L, 13 horses with babesiosis displayed high values
of the albumin concentration. Statistical data obtained
for the concentration albumin in Group II are shown in
table 3.
In this group, the -globulin fraction was also resolved
into two subfractions:
1
-globulin and
2
-globulin,
respectively. The
1
-globulin values from sick horses were
similar to the results obtained in healthy horses for the same
subfraction. Conversely, a statistically significant increase
(P < 0,005) in the median
2
-globulin sub-fraction was
observed in Group II (table 3). Fourteen of the 34 horses
studied, showed high values of
2
-globulin sub-fraction,
mainly 5 of them (figure 2).
The -globulin fraction in horses with babesiosis
was also resolved into two subfractions:
1
-globulin and
2
-globulin, respectively. Statistical results are shown in
table 3. The
1
and
2
-globulin median from sick horses
was similar to the results obtained in healthy horses for the
same subfraction. Although 3 horses in particular, had an
increase in the
2
-globulin and a decrease in
1
-globulin,
2 animals presented an increase in both sub-fractions and
9 horses showed a decrease in both sub-fractions.
The median value of the -globulins fraction obtained
in the sick horses (1.36 g/L) was significantly higher
(P < 0.01) than that obtained in Group I (table 3).
Finally, 12 of the sick animals displayed a ratio lower
than 1 and a low albumin concentration was observed in
the majority of them. The statistical results obtained for the
albumin/globulin ratio in Group II are shown in table 3.
DISCUSSION
Hyperproteinemia in babesiosis is usually caused by
dehydration, whose main cause is the letargy associated to
the disease (Divers 1998). In addition, the increase in the
-globulin concentration also contributes to the increase
in the total proteins concentration (Radostits et al 1999).
In our study, the median value obtained for the serum
total proteins in the Group II was significantly higher
(P < 0.01) than that obtained in the Group I (table 3).
Hypoproteinemia observed in six animals may be related
to the severe anorexia that characterizes the disease in its
acute form, as well as the feverish symptoms displayed
(table 1). In some cases, hypoproteinemia can be so severe
176
R BARRERA ET AL
proteins are usually evaluated together and reference is
made to the -globulin fraction without indicating the
sub-fraction (Sevelius and Andersson 1995, Kaneko
1997). The
1
-globulin median from the sick horses was
similar to the results obtained in the healthy horses for the
same subfraction (table 3). Changes of the
1
-globulin
concentration do not appear to be important from a
diagnostic point of view. However, the median value for
the
2
-globulin subfraction in Group II was significantly
higher (P < 0.05) when compared with that obtained in
Group I (table 3). The increase of the -globulin fraction
occurs frequently in equidae suffering from different
pathologies (Carapeto et al 2006). Although its increase has
not been described in equine babesiosis, a clear increase
in the
2
-globulin sub-fraction was observed in five of the
horses studied (figure 2). Although this increase is due to
tissue damage and inflammatory processes, elevation of
the -globulin fraction may be observed in diseases that
are not linked to inflammation, such as occurs in cases of
hepatic or renal damage (Kaneko 1997). This fact may
explain the results observed in some of the horses studied
because when a hepatopathy is developed, an increase in
the
1
-glycoprotein-acid,
1
-antitrypsin, haptoglobin and
2
-macroglobulin can be observed (Sevelius and Andersson
1995). In addition, animals with babesiosis frequently
develop renal disease resulting from dehydration and
haemoglobinuria by intra-vascular haemolysis produced
by the parasite (Navarrete y Serrano 1999). Finally, the
increase of -globulin fraction may be cut off by the
decrease in the concentration of the haptoglobin, usually
observed in animals with haemolytic anaemia, which
characterizes the disease (Jain 1993).
1
2
3
4
5
7
Figure 1. Densitometer scan of the serum electrophoresis of
a healthy horse. 1: albumin; 2:
1
-globulin; 3:
2
-globulin;
4:
1
-globulin; 5:
2
-globulin; 6: -globulins.
Lectura densitomtrica de la electroforesis srica de un ca-
ballo sano. 1: albmina; 2:
1
-globulina; 3:
2
-globulina; 4:
1
-globulina;
5:
2
-globulina; 6: -globulinas.
1
2
3
4 5
6
Figure 2. Densitometer scan of the serum electrophoresis of
a horse with babesiosis showing an increase of the
2
-globulin
subfraction. 1: albumin; 2:
1
-globulin; 3:
2
-globulin; 4:
1
-
globulin; 5:
2
-globulin; 6: -globulins.
Lectura densitomtrica de la electroforesis srica de un caballo
con babesiosis mostrando un aumento en la fraccin de
2
-globulinas.
1: albmina; 2:
1
-globulina; 3:
2
-globulina; 4:
1
-globulina; 5:
2
-
globulina; 6: -globulinas.
that oedema in the limbs of 3 horses was observed in our
study (table 1).
The median concentration of albumin in Group II
was higher than that obtained in Group I, although non-
statistically significant difference was observed (table 3).
The increase in the albumin concentration may be explained
as a consequence of dehydration because no pathological
processes with increase in the production of albumin have
been described (Kaneko 1997). In order to produce clinical
signs, it is considered that the serum albumin level must
decrease until some values below 15.0 g/L. The presence
of oedema was verified in the 3 horses that presented
such values. The decrease of the albumin concentration
in horses with babesiosis is related to the hepatopathy
that may develop during the disease (Colville 2002) and
to the symptomatic anorexic state (Stockham and Scott
2002) observed in 23 horses (table 1). Hypoalbuminemia
is usually related to chronic cases and a prolonged course.
Furthermore, the albumin concentration variation is usually
associated to that of serum total proteins since it is the most
abundant protein in the blood, as usually observed in the
animals studied. However, in 5 of the 14 horses that showed
an increase in serum total proteins, hyperalbuminemia
was not the main cause of the increase, it was a severe
hypergammaglobulinemia that had contributed noticeably
to that increase.
The -globulin fraction are known as acute stage
proteins because their concentration increases immediately
after an inflammation or a wound (Stockham and Scott
2002). In horses, the concentration of
1
-globulin is
lower than
2
-globulin (Kohn 1997), although functional
differences between them have not been reported. These
177
ELECTROPHORESIS, SERUM, BABESIOSIS, HORSE
When the results of -globulin sub-fractions in horses
with babesiosis were compared with those obtained in the
control group, no significant differences were observed
(table 3). Changes in this fraction depend on different factors,
such as the progress of the disease (acute or chronic) and
the course or severity of the haemolytic anaemia caused.
In addition, an increase in its concentration related to acute
hepatic damage has been described (Kaneko 1997) as a
consequence of the increase in the transferrin concentration.
This protein also contributes to the -globulin decrease
if the hepatic disease is chronic (Barta and Pourciau
1984). Conversely, horses suffering from babesiosis are
characterized by haemolytic anaemia. Although this has not
been confirmed in natural clinical processes, the -globulin
increase is a consequence of in vitro haemolysis caused by
the presence of free haemoglobin. In addition, a decrease
in the -globulin concentration as a result of a decrease
in the pepsin concentration in cases of clinical haemolysis
has been described (Barta and Pourciau 1984). Horses with
babesisosis occasionally show disseminated intra-vascular
coagulation (Radostits et al 1999) that causes an increase
in the plasminogen concentration, which is included in the
-globulin fraction. Finally, -globulin fraction concentration
may be related to the albumin fraction in cases of extra-
vascular liquid exudation. This fact was observed in two
of the horses that presented lower -globulin values and
hypoalbuminemia.
In babesiosis, the immunity mechanisms are mainly
of humoral nature (Morris et al 2002), although the
protozoa can activate a cellular-type immune response
(Tizard 1996). In this study, the median value obtained
for the -globulin fraction in sick horses was significantly
higher (P < 0.01) than the one obtained in healthy horses
(table 3). This increase observed in the serum -globulin
concentration (figure 3) is related to the humoral
immunity activated by the parasitation of the animals
studied. This occurs mainly in horses with a more severe
hypergammaglobulinemia and hepatic damage, although
the immune response in some animals may produce an
increase of immunoglobulin too small to be detected
through routine electrophoresis techniques (Jain 1993,
Stockham and Scott 2002).
Finally, although one third of the sick animals
displayed a albumin/globulin ratio lower than 1, the
absence of statistically significant difference against
healthy horses (table 3) may be due to the increase in
the albumin concentration observed in a high number
of animals (14 horses), as well as to the increase in the
Table 3. Median and interquartile range of the total protein concentration (g/L), serum protein fractions (g/L) and albumin/globulin
ratio (A/G) from Group I (healthy horses) and Group II (horses suffering from babesiosis). Statistical differences between both groups
are shown.
Mediana y rango intercuartlico de la concentracin de protenas totales (g/L), fracciones de protenas (g/L) y cociente albmina/globulina
(A/G) del Grupo I (caballos sanos) y del Grupo II (caballos con babesiosis). Se muestran las diferencias estadsticas obtenidas entre ambos grupos.
Total
protein
Albumin
1
-
globulin
2
-
globulin
1
-
globulin
2
-
globulin
-
globulin
A/G
GROUP I
(N = 19)
Median 6.00 3.21 0.12 0.53 0.65 0.41 0.94 1.05
IQR 0.60 0.44 0.05 0.23 0.25 0.42 0.29 0.35
GROUP II
(N = 34)
Median 7.18 3.63 0.12 0.68 0.69 0.36 1.36 1.22
IQR 3.23 1.65 0.08 0.33 0.35 0.42 1.22 0.76
Significance level P < 0.01 NS NS P < 0.05 NS NS P < 0.01 NS
IQR = interquartile range.
NS = non-statistically significant.
NS = no estadsticamente significativo.
1
2
3
4
5
6
Figure 3. Densitometer scan of the serum electrophoresis of
a horse with babesiosis showing an increase of the -globulin
fraction. 1: albumin; 2:
1
-globulin; 3:
2
-globulin; 4:
1
-globulin;
5:
2
-globulin; 6: -globulins.
Lectura densitomtrica de la electroforesis srica de
un caballo con babesiosis mostrando un aumento en la fraccin de
-globulinas. 1: albmina; 2:
1
-globulina; 3:
2
-globulina; 4:
1
-globulina;
5:
2
-globulina; 6: -globulinas.
178
R BARRERA ET AL
concentrations of
2
-globulin and -globulin fractions
discussed previously.
Results obtained in this study allow to conclude that
the increase of the -globulin fraction, associated in
some cases to the increase of the
2
-globulin fraction,
is a common finding in the serum electrophoresis of the
horses suffering from babesiosis and it can be very useful
for the diagnosis of the disease.
SUMMARY
Serum electrophoresis in cellulose acetate strips is a technique
commonly used to separate the protein fractions. This technique allows
detecting quantitative and qualitative changes in the serum proteins
associated with different diseases. The aim of this study was to characterize
the changes of the serum protein fractions produced in horses suffering
from babesiosis and to evaluate its application in its diagnosis. Serum
total proteins were calculated and the serum electrophoresis from 53
horses was performed. Animals were classified in two groups: Group I
(19 healthy horses) and Group II (34 horses suffering from babesiosis).
Babesiosis was diagnosed by indirect immunofluorescence antibody
assay. Data obtained in the group of sick horses were compared with
those from the healthy horses. Median values from total proteins and
-globulins fraction were significantly higher (P < 0.01) than that observed
for the control group. In addition, a statistically significant increase was
observed (P < 0.05) in the values of
2
-globulin subfraction in the sick
horses. Results obtained in this study allow concluding that the most
significant alteration observed in the serum electrophoretic pattern of
horses suffering from babesiosis is the increase of
2
-globulin and
-globulin fractions.
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179
Arch Med Vet 42, 179-186 (2010)
ARTCULO ORIGINAL
Aceptado: 03.03.2010.
# Proyecto FONDEF DO6I1020. Casilla 15-D Temuco, Chile;
oberrios@uct.cl
Almacenamiento en fro de espermatozoides de trucha arcoiris
(Oncorhynchus mykiss): Efectos en la motilidad, superxido intracelular, integridad
de la membrana plasmtica y potencial de membrana mitocondrial
#
Cold storage of sperm of rainbow trout (oncorhynchus mykiss): Effect on motility, intracellular
superoxide, plasma membrane integrity and mitochondrial membrane potential
O Berros
*
, I Valdebenito, F Treuln, A Ubilla
Escuela de Acuicultura, Universidad Catlica de Temuco, Temuco, Chile.
SUMMARY
In teleostei, as opposed to what happens in mammals, most of the studies that evaluate the quality storages of semen are oriented toward the exposure
of spermatozoa to some reactive oxygen species (ROS), the utilization of antioxidants in the diet, or the incorporation of these in seminal plasma. There
is no available the literature covering the presence of superoxide ions (O
2
._
), or the function of these on the interior of the spermatozoa that have been
stored. In this study, we evaluated the effect of storage on intracellular O
2
._
, motility, plasmatic membrane integrity, and mitochondrial membrane potential
(
Mit
) of spermatozoa of rainbow trout (oncorhynchus mykiss). Semen was extracted and put into storage for 12 days at 4 C. Spermatozoa motility,
(
Mit
), plasmatic membrane integrity were evaluated every 4 days, and intracellular O
2
._
was detected. It was found that 82.59% of the cells were positive
for the staining of O
2
._
on the day of sample extraction, while sperm motility,
Mit
and plasmatic membrane integrity only showed deterioration after
the eighth day of storage. Only
Mit
is correlated negatively with O
2
._
starting from the eighth day of storage (r = 0.56 P < 0.05). Regarding motility
and plasmatic membrane integrity, these were not affected by intracellular O
2
._
, although the deterioration observed in these last two indicators, could
have been caused by the prolonged contact of the spermatozoa with contaminating agents that were not evaluated in this study.
Palabras clave: almacenamiento, superxido intracelular, trucha arcoiris, espermatozoides.
Key words: storage, intracellular superoxide, rainbow trout, spermatozoa.
INTRODUCCIN
Es habitual en especies de cultivo utilizar el almace-
namiento de semen fuera del ambiente testicular, en caso
de diagnstico ictiosanitario o por ubicacin de los repro-
ductores a grandes distancias del sitio en que se realiza la
incubacin. Generalmente, este procedimiento es fcil de
ejecutar y no requiere de implementacin costosa ni de
conocimientos avanzados, por lo que frecuentemente se
practica en centros de cultivo.
Una forma de evitar el deterioro de los espermatozoi-
des almacenados es diluir el semen en soluciones salinas
isotnicas o diluyentes que imitan el medio en que se
encuentran dentro del testculo. Los beneficios de estas
soluciones estn centrados principalmente en mantener las
condiciones de humedad y control bacteriano del semen,
permitiendo conservar las potencialidades fecundantes
de los espermatozoides por largos periodos de tiempo
(Babiak y col 2006).
En atencin a lo anterior, se han identificado algunos
agentes que perjudican la viabilidad y motilidad de los
espermatozoides almacenados in vitro, entre estos se
cuentan las condiciones propias de los reproductores como
la edad de maduracin, o factores relacionados con el
medio de almacenamiento como temperatura, exposicin
a CO
2
, pH, concentracin de oxgeno o contaminacin
con orina (Billard y col 1993, Billard y col 1997, Ohta y
Izawa 1996, Bencic y col 2001, Ingermann y col 2002,
Liley y col 2002, Valdebenito y col 2009).
Los estudios relacionados con calidad del semen
de telesteos almacenado in vitro en su mayora estn
orientados a evaluar algunas caractersticas como color,
consistencia (Estay y Tllez 1994), densidad espermtica,
motilidad (Billard 1988, Aas y col 1991, Estay y Tllez
1994) o composicin del plasma seminal (Lahnsteiner y
col 1998) y, a diferencia de lo que ocurre con mamferos,
poco se ha estudiado el deterioro estructural o produccin
de especies reactivas de oxgeno (ROS) en espermatozoides
mantenidos fuera del testculo.
En relacin a lo anterior, en mamferos se ha observado
un evidente incremento en la concentracin de especies
reactivas del oxgeno (ROS), como resultado de patolo-
gas o por criopreservacin de las clulas espermticas
(Chatterjee y col
2001, Agarwal 2004).
180
O BERROS Y COL
Las ROS son una serie de radicales con carga negativa,
y por ser altamente inestables tienden a reaccionar con otras
molculas dentro de la clula, las ms frecuentes son el anin
superxido (O
2
.
), radical peroxilo (ROO
) y anin hidroxilo
(OH
Research Laboratories,
AK-116), cuyo colorante activo es 5,5,6,6-tetrachloro-
1,1,3,3-tetraethyl-benzamidazolocarbocyanin iodide (JC-1).
ste posee una estructura de carga positiva y lipoflica que
le permite ingresar fcilmente a las clulas y mitocondrias.
La carga negativa de la membrana mitocondrial interna pro-
duce agregacin del colorante JC-1, el cual forma dmeros
que emiten fluorescencia de color rojo cuando existe un
alto potencial mitocondrial. Cuando en la clula el
Mit
disminuye, el reactivo ingresado asume una forma monom-
rica que emite fluorescencia verde (Marchetti y col 2004).
Ambos colores son detectados por citometra.
Para la experimentacin se adicionaron 3 x 10
6
esperma-
tozoides a 1 ml de diluyente, la suspensin se centrifug a
470 g por 5 minutos y se elimin el sobrenadante. Luego, el
pellet fue disuelto en una concentracin final de 10 g JC-1/
ml diluyente. Para que actuara el colorante la suspensin se
incub 15 minutos a 4 C protegido de la luz, y se centrifug
nuevamente a 470 g por 5 minutos. Se elimin el sobrenadante
y los espermatozoides contenidos en el pellet fueron disueltos
en 400 l de diluyente para ser evaluados al citmetro.
EVALUACIN DE INTEGRIDAD DE MEMBRANA PLASMTICA
La integridad de la membrana plasmtica se evalu
con el kit Live/Dead
n
d
e
l
a
m
o
t
i
l
i
d
a
d
(
m
i
n
)
1 4 8 2
(Das)
Figura 1. Mantencin de la motilidad de espermatozoides de
trucha arcoiris (o. mykiss) almacenados in vitro. Valores pro-
medio DE.
Maintenance of motility of spermatozoa of rainbow trout
(o. mykiss) stored in vitro. Mean values SD.
182
O BERROS Y COL
de los espermatozoides (figura 2). El anlisis de varianza
ANDEVA mostr diferencias significativas en el porcentaje
de espermatozoides que presentaron O
2
._
solo el ltimo
da de evaluacin (P < 0,05), no obstante, se encontr alta
correlacin entre los das de almacenamiento del semen
y el porcentaje de espermatozoides que presentaba O
2
._
(r = 0,8 P < 0,05).
Los grficos de dispersin de la figura 3 muestran
en la regin superior izquierda la poblacin de aquellos
espermatozoides que se tieron positivamente con DHE.
Se observa que este grupo de clulas se mantuvo ms o
menos constante a partir del primer da de evaluacin.
INTEGRIDAD DE MEMBRANA CITOPLASMTICA
La integridad de la membrana plasmtica tambin se vio
afectada con el almacenamiento del semen; al respecto, se
encontr que el 88,6 9,3% de los espermatozoides regis-
trados en el citmetro presenta la membrana ntegra el da
de extraccin (figura 2). Este porcentaje se ve disminuido
a la mitad, 50,3 14,9%, el octavo da de almacenamiento;
y despus de doce das de encontrarse los espermatozoi-
des fuera del testculo se observa el 47,0 13,7% de los
espermatozoides con la membrana intacta, caracterizada
por la fluorescencia de color verde. Al igual que el
Mit
,
el anlisis de varianza ANDEVA muestra diferencias
significativas de la integridad, a partir del octavo da de
almacenamiento (P < 0,05). Correlacionando los das
de almacenamiento con la integridad de la membrana, se
encontr que la permanencia de los espermatozoides fuera
del testculo la afecta negativamente (r = 0,8 P < 0,05).
Al correlacionar el O
2
._
con la integridad, para observar
algn grado de relacin, no se encuentra correlacin entre
ambos (P > 0,05).
El grfico de dispersin muestra el desplazamiento de
la poblacin celular desde el cuadrante inferior derecho de
clulas con la membrana ntegra hacia el cuadrante superior
derecho, donde se detectan las clulas con la membrana
deteriorada o muertas, teidas con IP (figura 4).
POTENCIAL DE MEMBRANA MITOCONDRIAL
Se observ gran porcentaje de espermatozoides con
alto
Mit
el primer da de evaluacin (80,8 11,6%), dis-
minuyendo a la mitad los espermatozoides con alto
Mit
el octavo da (53,8 20,3%). Finalmente la evaluacin
correspondiente al duodcimo da revel un 44,9 26,1%
de espermatozoides con alto
Mit
(figura 2). Por otro
lado, el test de comparaciones mltiples (T-Tukey) muestra
diferencias significativas del
Mit
entre los das 1 y 4
0
10
20
30
40
50
60
70
80
90
100
E
s
p
e
r
m
a
t
o
z
o
i
d
e
s
(
%
)
Mit
Integr. membrana plasmtica
O
2
.
r = 0.6 r = 0.8 r = 0.8
a
a
a
a
a
a
a
b
b
b
b
b
Da 1 Da 4 Da 8 Da 12
Figura 2. Porcentaje de espermatozoides de trucha arcoiris (o. mykiss) con
Mit
alto, membrana citoplasmtica ntegra y con superxido
intracelular. En la parte superior se exhibe el coeficiente de correlacin con respecto a los das de almacenamiento y la significancia,
aqu letras diferentes muestran diferencias estadsticamente significativas (p < 0,05). Los resultados se dan en promedio DE. El
recuadro muestra los das de las evaluaciones.
Percentage of spermatozoa of rainbow trout (o. mykiss) with high
Mit
, cytoplasmic integral membrane and with intracellular superoxide.
At the top is displayed the correlation coefficient with respect to days of storage, and the significance, here different letters show statistically significant
differences (p < 0.05). The results are average SD. The box shows the days of the evaluations.
183
ALMACENAMIENTO, SUPERXIDO INTRACELULAR, TRUCHA ARCOIRIS, ESPERMATOZOIDES
humanos y equino sometidos a criopreservacin (De Iuliis
y col 2006, Burnaugh y col 2007).
En espermatozoides de mamferos, el O
2
._
intracelular
es consecuencia de los procesos metablicos que habi-
tualmente ocurren en la mitocondria (de Lamirande 1997,
Sanocka y Kurpisz 2004); aqu este radical es generado y
liberado al citoplasma donde es transformado a H
2
O
2
, el
cual finalmente difunde hacia el espacio extracelular (de
Lamirande 1997, Koppers y col 2008).
En humanos, habitualmente es baja la concentracin
de O
2
._
disponible al interior de la clula, el cual formara
parte de una cascada de eventos que finalizara con la
capacitacin espermtica (de Lamirande 1998).
No se encuentran trabajos disponibles que traten la
produccin de superxido intracelular y la funcin que
ste cumple al interior del espermatozoide de peces.
Los escritos publicados estn orientados a la utilizacin
de antioxidantes en la dieta, su aplicacin en el plasma
seminal o la exposicin de los espermatozoides a otros
tipos de ROS, donde se pone nfasis en la evaluacin
de la motilidad, capacidad fecundante, lipoperoxidacin
y dao del ADN de los espermatozoides por efecto de
antioxidantes (Ciereszko y Dabrowski 1995, Kiron y
col 2004, Mansour y col 2006, Zhou y col 2006, Canyurt
y Akhan 2008).
D
D
H
E
A B
C
Figura 3. Comportamiento de los espermatozoides de trucha
arcoiris (o. mykiss) respecto de la tincin de DHE para supe-
rxido. Se observa a la mayora de los espermatozoides ubicados
en el cuadro superior izquierdo, que corresponde a las clulas
con O
2
._
intracelular, durante todos los das que permanecieron
almacenadas. A = da 1, B = da 4, C = da 8 y D = da 12.
Behavior of spermatozoa of rainbow trout (o. mykiss) after
DHE staining for superoxide. Note the majority of sperm located in the
upper left box, which corresponds to the cells with intracellular O
2
._
,
every day they remained in storage. A = 1st, B = 4 days, C8 days and
D = 12 days.
I
P
SYBR-14
A B
C D
Figura 4. Tincin de SYB14 - IP para integridad de membrana
plasmtica de trucha arcoiris. Se observa el desplazamiento
de los espermatozoides desde el cuadro inferior derecho, que
corresponde a la membrana ntegra, hacia el cuadro superior
derecho correspondiente a la tincin de ioduro de Propidio, este
ltimo ti las clulas muertas. A = da 1, B = da 4, C = da 8
y D = da 12.
Staining SYB14 - IP for plasma membrane integrity of
rainbow trout. It shows the movement of sperm from the lower right
square that corresponds to the integral membrane, towards right upper
square for the propidium iodide staining, the latter stained dead cells.
A = 1st, B = 4 days, C8 days and D = 12 days.
con los das 8 y 9 (P < 0,05). Al correlacionar los das de
almacenamiento versus el porcentaje de espermatozoides
con alto
Mit
se encuentra correlacin negativa entre
ambos (r = 0,6 P < 0,05), al igual que al correlacionar
el porcentaje de clulas con tincin positiva para O
2
._
y
Mit
, donde tambin se obtiene un ndice de correlacin
negativo r = 0,56 P < 0,05.
El test de comparaciones mltiples entre el porcentaje
de espermatozoides con tincin positiva para O
2
._
y el
porcentaje de espermatozoides con alto
Mit
nuevamente
muestra diferencias significativas entre los das 1 y 4 con
los das 8 y 12.
En el grfico de dispersin de la figura 5 se aprecia un
desplazamiento evidente de las clulas desde el cuadrante
inferior derecho, donde se encuentran los espermatozoides
con alto
Mit
, hacia el cuadrante superior derecho donde
se encuentran las clulas con bajo
Mit
, este ltimo grupo
aumenta notoriamente los das 8 y 12.
DISCUSIN
El tiempo que permanece almacenado el semen fuera
del ambiente testicular produce efectos perjudiciales en
los espermatozoides. La presencia del anin superxido
(O
2
._
) intracelular, una especie reactiva de oxgeno (ROS),
anteriormente haba sido detectado en espermatozoides
184
O BERROS Y COL
peces (Alavi y col 2004, Alavi y Cosson 2005, Alavi y
col 2007). Una particularidad que tienen los espermato-
zoides de salmnidos, entre los que se encuentra la trucha
arcoiris, es la carencia de movimiento cuando se hallan
dentro del testculo o en el lquido seminal, caracterstica
que permite mantenerlos inactivos in vitro, recurriendo a
soluciones isoosmticas con una concentracin de potasio
similar a la del semen, puesto que este ion es el principal
responsable de inhibir el movimiento dentro del testculo
(Morisawa y Morisawa 1986). Inversamente, cuando se
requiere evaluar motilidad, los espermatozoides deben
ser activados depositndolos en agua o medios salinos
isoosmticos, con una concentracin de potasio inferior
al lquido seminal, donde, al igual que en la naturaleza,
el movimiento finalizar 15 a 30 s despus que la clula
haya sido activada (Christen y col 1987).
En este estudio, el movimiento espermtico present
un evidente descenso a partir del octavo da, observndose
solo la mitad de los espermatozoides con movimiento en
esta evaluacin. Considerando el largo periodo de tiempo
transcurrido antes de que la motilidad fuera afectada, al
parecer sta no estuvo influenciada negativamente por los
radicales O
2
._
intracelulares, y el perjuicio podra deberse
a otros factores externos que no fueron evaluados en este
trabajo, como puede ser la contaminacin con orina o mi-
croorganismos (Dreanno y col 1998, Ochsendorf 1999).
Los espermatozoides obtienen la energa para el
movimiento del flagelo a partir del adenosin-trifosfato
(ATP) (Christen y col 1987). La forma como adquieren
esta molcula puede variar entre las diferentes especies,
as en Cyprinus carpio o carpa comn, por ejemplo,
los espermatozoides obtienen parte de la energa para
el movimiento por va glicoltica, en trucha arcoiris, en
cambio, obtiene prcticamente la totalidad de la energa
a partir de respiracin aerbica, con participacin de las
mitocondrias, las cuales generan ms O
2
._
a medida que
aumenta el movimiento flagelar (Zietara y col 2009).
En este aspecto, llama la atencin el alto porcentaje de
clulas que presentaron tincin positiva para O
2
._
, aun
cuando durante los ensayos los espermatozoides perma-
necieron en soluciones que no activaron su motilidad, lo
cual debera disminuir considerablemente la generacin
de este radical en las mitocondrias. Lo anterior hace
presumir que los O
2
._
observados en gran porcentaje de
espermatozoides podran provenir de otro compartimento
distinto a las mitocondrias (De Iuliis y col 2006) o tener
un origen extracelular como se ha observado en algunos
mamferos (Marklund 1984).
La importancia de la membrana plasmtica radica
principalmente en funciones de transporte, reconocimiento
de seales, balance hdrico y capacidad fecundante de
la clula espermtica (Boitano y Omoto 1991, Cabrita
y col 2001). En este estudio, se entregan los primeros
antecedentes sobre integridad de membrana en esperma-
tozoides de trucha arcoiris almacenados. No obstante, en
espermatozoides de mamferos sometidos a criopreservacin
ha sido ampliamente estudiada (De Baulny y col 1997,
Rasul y Anzar 2001).
I
n
t
e
n
s
i
d
a
d
Intensidad
Alto
MIT
(uorescencia roja)
Bajo
MIT
(uorescencia roja)
A B
C D
Figura 5. Comportamiento del
Mit
durante el almacenamien-
to. Se observa la mayora de los espermatozoides en el cuadro
inferior derecho, indicando un alto
Mit
los primeros das de
evaluacin, mientras que en las dos ltimas evaluaciones se
produjo desplazamiento de la mancha hacia la parte superior
derecha donde se encuentran los espermatozoides con bajo
Mit
,
caracterizados por una mezcla de ambos colores. Las flechas de
los ejes indican intensidad de la fluorescencia. A = da 1, B = da
4, C = da 8 y D = da 12.
Mit
behavior during storage, observed the majority of
sperm in the lower right square indicating a high
Mit
first days of
evaluation, while in the last two assessments were moving toward the part
upper right in which are spermatozoa with low
Mit
, characterized by a
mixture of both colors. The arrows indicate the axes of the fluorescence
intensity. A = day 1, B = day 4, C = day 8 and D = day 12.
A diferencia de lo observado en espermatozoides de
humanos y jabal (Guthrie y Welch 2006), en el presente
estudio se detect O
2
._
intracelular en gran porcentaje de
los espermatozoides desde el da de extraccin del semen.
Sin embargo, no se advirti que este radical afectara la
motilidad, integridad de la membrana plasmtica o el
Mit
los primeros siete das de almacenamiento. Lo anterior
podra indicar la inocuidad de los radicales superxido
cuando se encuentran al interior celular, al igual que lo
observado en espermatozoides incubados en medios con
xantina-xantina oxidasa (XA-XO), donde el dao a la
motilidad y estructuras celulares es producido principal-
mente por el exceso de H
2
O
2
liberado del metabolismo de
los O
2
._
y no directamente por la presencia de este radical
(Aitken y col 1993, Baumber y col 2000, Guthrie y Welch
2006, Martnez-Pastor y col 2009).
La motilidad espermtica es un indicador que habi-
tualmente se evala en los experimentos con semen de
185
ALMACENAMIENTO, SUPERXIDO INTRACELULAR, TRUCHA ARCOIRIS, ESPERMATOZOIDES
Una caracterstica de la membrana plasmtica de
trucha arcoiris es su alto contenido en colesterol (Pustowka
2000), lo cual permite a la clula regular funciones como
la fluidez y permeabilidad (Sanocka y Kurpisz 2004). En
este trabajo no se observ relacin entre la integridad de
la membrana y O
2
._
presente en el interior celular, ya que
al correlacionar ambos no hubo asociacin. Sin embargo,
cuando se correlacion integridad con das de almacena-
miento s se encontr elevada correlacin negativa, r = 0,8,
lo que podra indicar perjuicio del almacenamiento, tal vez
debido a otros radicales generadores de oxgeno, como
por ejemplo, perxido de hidrgeno generado a partir de
O
2
._
al transcurrir los das de almacenamiento o por otros
agentes contaminantes, ya por un tema de proteccin
al medio ambiente; la solucin salina isoosmtica que
se utiliz para la conservacin de los espermatozoides
careca de cualquier tipo de antibitico, razn por la cual
es muy probable que la muestra de semen haya contado
con una gran carga de microorganismos, algunos de los
cuales podran haber producido perxido de hidrgeno
(Ochsendorf 1999), daando la membrana y alterando la
motilidad espermtica durante el almacenamiento.
Otro agente que frecuentemente se puede encontrar en
la extraccin del semen es la orina, y aunque se tomaron
algunas precauciones para evitar su extraccin junto con la
muestra de semen, probablemente stas no fueron suficientes.
La orina en el semen altera la molaridad y concentracin
de potasio, adems influye en la concentracin de ATP y
motilidad espermtica (Dreanno y col 1998).
El
Mit
es el nico indicador que fue afectado por
O
2
._
intracelular, lo cual se demuestra en la correlacin
negativa obtenida (r = 0,56) al asociar ambos indicadores.
De acuerdo a los resultados, los espermatozoides tambin
fueron afectados por los das de almacenamiento, presentan-
do una disminucin en el potencial a partir del octavo da.
Esto ltimo probablemente debido a la interaccin directa
y prolongada entre O
2
._
y algunas molculas que son pro-
ductos de la cadena respiratoria en la mitocondria (Koppers
y col 2008), ya que, a diferencia del ROS intracelular, el
extracelular (principalmente perxido de hidrgeno) no
produce dao peroxidativo a corto plazo en la membrana,
ni afecta el
Mit
(Baumber y col 2000), inversamente a lo
encontrado en espermatozoides de jabal, donde el exceso
de O
2
._
s provoca disminucin del potencial mitocondrial y
motilidad, a pesar de encontrarse este radical en un escaso
nmero de espermatozoides (Guthrie y Welch 2006).
En conclusin, en nuestro estudio se observ un alto por-
centaje de espermatozoides que presentaron O
2
._
intracelular.
De acuerdo a los anlisis estadsticos, este anin no afect
la motilidad ni la integridad de la membrana plasmtica.
Sin embargo, el potencial de membrana mitocondrial s es
afectado, lo cual podra deberse a la exposicin directa y
prolongada de O
2
._
con molculas generadas como producto
de la respiracin dentro de la mitocondria.
Por otro lado, teniendo en cuenta los das de almacena-
miento, se observ deterioro en todos los indicadores aqu
evaluados, no obstante, dos de ellos (motilidad e integridad
de la membrana citoplasmtica) no son efecto directo de los
O
2
._
, sino que al parecer de otros factores no evaluados en
esta investigacin, como puede ser el perxido de hidrge-
no generado a partir de la contaminacin bacteriana o por
efecto de la contaminacin involuntaria con orina.
Finalmente, considerando la importancia que tiene la
reproduccin de peces en cautiverio y silvestres, resulta
conveniente profundizar la investigacin en aspectos
celulares y as proporcionar informacin til para la ela-
boracin de medios de cultivos y otros insumos utilizados
en el manejo de gametos.
RESUMEN
A diferencia de lo que ocurre en mamferos, en telesteos la mayora de
los estudios que evalan la calidad del semen almacenado estn orientados a
la exposicin de los espermatozoides a algunas especies reactivas de oxgeno
(ROS), a la incorporacin de antioxidantes en la dieta o a la aplicacin de
stos en el plasma seminal. No se encuentran trabajos disponibles que traten
la presencia del radical superxido (O
2
._
), ni la funcin que ste cumple
al interior del espermatozoide cuando se encuentran almacenados. En la
presente investigacin se evalu el efecto del almacenamiento en el O
2
._
,
motilidad, integridad de la membrana plasmtica y potencial de membrana
mitocondrial (
Mit
) en espermatozoides de trucha arcoiris (oncorhynchus
mykiss). Para ello se extrajo el semen, el cual fue almacenado durante
12 das a 4 C. Cada 4 das se evalu motilidad,
Mit
, integridad de la
membrana plasmtica y se detect O
2
._
intracelular en los espermatozoides.
Se encontr un 82,59% de clulas con tincin positiva para O
2
._
el da de
extraccin de la muestra, mientras que la motilidad,
Mit
y la integridad
de la membrana plasmtica, solo mostraron deterioro despus del octavo da
de almacenamiento. nicamente el
Mit
se correlaciona negativamente
con O
2
._
a partir del octavo da de almacenamiento (r = 0,56 P < 0,05).
Respecto a la motilidad e integridad de la membrana plasmtica no fueron
afectadas por el O
2
._
intracelular, no obstante el deterioro observado en
estos dos ltimos indicadores podra deberse al contacto prolongado
de los espermatozoides con agentes contaminantes no evaluados en el
presente trabajo.
AGRADECIMIENTOS
A la Direccin General de Investigacin de la Universidad Catlica
de Temuco, por su colaboracin.
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Arch Med Vet 42, 187-193 (2010)
ORIGINAL ARTICLE
Accepted: 06.08.2010.
* cristinagatica@gmail.com
Effects of crowding on blood constituents and flesh quality
variables in Atlantic salmon (Salmo salar L.)
Efecto del confinamiento sobre las variables sanguneas y
calidad de carne de Salmn Atlntico (Salmo salar L.)
MC Gatica
a*
, GE Monti
b
, TG Knowles
c
, CB Gallo
a
a
Instituto de Ciencia Animal y Tecnologa de Carnes, Facultad de Ciencias Veterinarias,
Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
b
Instituto de Medicina Preventiva Veterinaria, Facultad de Ciencias Veterinarias,
Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
c
School of Clinical Veterinary Science, University of Bristol, Langford, Bristol, UK.
RESUMEN
Se compararon en los das 0, 1, 4, 7 y 10 post mortem, en trminos de variables de la calidad de la carne y constituyentes sanguneos, los efectos
de la anestesia (como tratamiento del control) y el confinamiento controlado con disminucin del nivel de oxgeno en salmones tamao cosecha (Salmo
salar). Los peces fueron mantenidos en triplicado en estanques y muestreados despus de anestesiar con AQUI-S
1
]
+
_
,
+ 1 86
18 2..8
_
,
(Holmes 1962)
Cortisol concentration was determined by a RIA
technique (Radio Immune Assay). The samples were
measured on a radioimmunoassay using marked cortisol
with iodine 125 and this marked hormone is made to
compete with cold hormone, which is in the samples.
As the antibody is attached to the tube, this test is
called coat-to-count. The antibody is highly specific
for cortisol and has very low reaction crossed with
other steroids such as cortisone and corticosterone. At
FISENLAB laboratory all RIAs have been validated
for use in animals.
MUSCLE PH, COLOUR, ASTHAXANTIN CONTENT
AND PERCENTAGE WEIGHT LOSS
Muscle pH and colour scores were determined at 0
(1 hour post-mortem), 1, 4, 7 and 10 days post-mortem.
Muscle pH was measured by directly inserting a pH probe
(Checker 200) in the left NQC fillet of each fish; probe
insertion was always performed at the same position
and by the same person. At the same time, visual colour
scores were obtained using the DSM SalmoFan (DSM
Nutritional Products, Chile), which uses a scale ranging
from 21 (light red) to 34 (dark red), under standardised
illumination in a Salmon Colour Box and by the same
person every time. Asthaxantin content was determined
by HPLC on days 1, 4, 7 and 10 postmortem, also in the
left NQC fillet. Between measurements the fillets were
kept refrigerated (0-4 C) in individually labelled plastic
bags. The right NQC fillets were weighed and also kept
individually labelled in refrigeration (0-4 C); any liquid
exudate present in the bag was wiped off before each
weighing. Weight loss was determined as the difference
between day 1 and days 4, 7, 10, and then calculated as a
percentage of the initial weight on day 1. All flesh quality
analysis were performed at TraceLab Analytical Services,
Puerto Montt, Chile.
STATISTICAL ANALYSIS
The normality of the distribution of the blood variables
was determined by visual inspection of a histogram and by
means of the Shapiro-Wilks Normality Test. A two sample
t-test was used to test variables with a normal distribution
and the Wilcoxon Rank Sum Test for those not normally
distributed. Flesh quality data were repeated measures
and were analysed with a model that allowed a varying
intercept (Singer and Willett 2003), expressed as:
Y
ij
= b
0ij
+
1
Days post mortem0
j
+
3
Days post
mortem10
j
+
4
Treatment
j
b
0ij
=
0i
+
0j
+
0ij
Where i = days postmortem and j = individual
This model assumes that
0j
takes a particular variance
structure (simple, compound symmetry first-order
autoregressive and unstructured (more details in the next
paragraph) for the repeated measures within-individuals
and the error term
0ij
is a Gaussian random variable that
are uncorrelated and have expectations 0 and and variances
(N (0,
) (Singer 1998).
With typical repeated measures, as here, two measure-
ments that are taken at adjacent time are more correlated
than those measurements taken several time points apart
(Little et al 1996). Therefore, it is important to account
for these correlations in estimating the effects of time and
of treatment.
Several error structures were evaluated including
simple (no correlation), compound symmetry, first-order
autoregressive and unstructured (estimating a correlation
for each separate correlation). The different correlation
structures were evaluated using Akaikes Information
Criterion (AIC). AIC can be used to compare models
with the same fixed effects but that differ in the variance
structure (Little et al 1996). We found the first-order
autoregressive (AR(1)) correlation structure to provide
the best fit to these data. For the first-order autoregressive
model, the correlations decrease exponentially with the
distance between measurements. Therefore, we illustrate
below a correlation sub-matrix for the ith individual:
1
1 2
2 1
3 2 1
2 3