Flow cytometric assessment of fresh and frozen-thawed Canada goose (Branta canadensis) semen
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Theriogenology 76 (2011) 843– 850
www.theriojournal.com
Flow cytometric assessment of fresh and frozen-thawed Canada
goose (Branta canadensis) semen
Agnieszka Partykaa,b,*, Ewa Łukaszewiczb, Wojciech Niżańskia
a
Wrocław University of Environmental and Life Sciences, Faculty of Veterinary Medicine, Department of Reproduction and Clinic of Farm
Animals, Wrocław, Poland
b
Wrocław University of Environmental and Life Sciences, Faculty of Biology and Animal Breeding, Department of Poultry Breeding,
Wrocław, Poland
Received 10 February 2011; received in revised form 9 April 2011; accepted 18 April 2011
Abstract
The present study was conducted to investigate spermatozoal membrane integrity, acrosome integrity, mitochondrial activity,
and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen with the use of the flow cytometry.
The experiment was carried out on ten, 2-year-old, Canada goose ganders. The semen was collected twice a week, by a
dorso-abdominal massage method, then pooled and subjected to cryopreservation in straws, in a programmable freezing unit with
the use of dimethyloformamide (DMF) as a cryoprotectant. Frozen samples were thawed in a water bath at 60 °C. The freezing
procedure was performed ten times. For the cytometric analysis the fresh and the frozen-thawed semen was extended with EK
extender to a final concentration of 50 million spermatozoa per mL. Sperm membrane integrity was assessed with SYBR-14 and
propidium iodide (PI), acrosomal damage was evaluated with the use of PNA-Alexa Fluor®488 conjugate, mitochondrial activity
was estimated with Rhodamine 123 (R123), and spermatozoal DNA integrity was measured by the sperm chromatin structure
assay (SCSA). The cryopreservation of Canada goose semen significantly decreased the percentage of live cells, from 76.3 to
50.4% (P ⬍ 0.01). Moreover, we observed the significant decrease in the percentage of live spermatozoa with intact acrosomes
(P ⬍ 0.01), but we did not detect significant changes in the percentage of live spermatozoa with ruptured acrosomes. However,
after thawing 50% of Canada goose live spermatozoa retained intact acrosomes. Furthermore, the percentage of live spermatozoa
with active mitochondria was significantly lower in the frozen-thawed semen than in the fresh semen (P ⬍ 0.05). Nevertheless,
after thawing the mitochondria remained active in almost 50% of live cells. In the present study, we observed no changes in the
percentage of sperm with fragmented DNA after freezing-thawing of Canada goose semen. In conclusion, the present study
indicates that even the fresh Branta canadensis semen might have poor quality, the cryopreservation of its semen did not provoke
spermatozoal DNA defragmentation and half of the spermatozoa retained intact acrosomes and active mitochondria after
freezing-thawing.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Avian semen; Goose semen; Cryopreservation; Semen evaluation; Flow cytometry
1. Introduction
In goose breeding, artificial insemination is not as
* Corresponding author. Tel.: ⫹48 71 32 05 300; fax: ⫹48 71 32 commonly used as in turkeys or chickens. Compared to
01 006. mammalian or chicken semen, the data on freezing
E-mail address: partykaagnieszka@gmail.com (A. Partyka). gander semen are rather limited. This is partly caused
0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.04.016844 A. Partyka et al. / Theriogenology 76 (2011) 843– 850
by the low reproductive capability, resulting from the 2.2. Semen collection procedure
poor semen quality, low egg production, fertility, and
Semen of ten males was collected two times a week
hatchability rates that characterized geese in compari-
by the dorso-abdominal massage method [7] and then
son to other poultry species. Moreover, geese have a
pooled to obtain a sufficient sample for analysis. Dur-
relatively short reproductive period and ganders pro-
ing the semen collection particular care was taken to
duce a small volume of ejaculate (0.05–1.0 mL) with a
minimize the contamination of semen with uric acid or
low spermatozoa concentration (0.03– 0.8 ⫻ 109/mL)
feces. After collection the pool was split into two ali-
and a low number of live normal cells (10 – 60%) [1].
quots, one for fresh semen evaluation and the other one
However, the possibility of semen storage in the liquid
for the cryopreservation procedure. During one repro-
nitrogen for an unlimited period may be helpful in wild
ductive cycle, 10 semen collections, from each male,
goose gene pool preservation.
were performed.
It is well acknowledged, in practice, that one of the
criteria to predict the freezability of spermatozoa is the 2.3. Semen cryopreservation
quality of the fresh semen. Cryopreservation induces
many unfavorable changes in spermatozoa that may Within 20 min following the collection, semen sam-
lead to cell injury and cause lower quality of frozen- ples were subjected to cryopreservation in accordance
thawed semen in comparison with a fresh ejaculate. with the procedure of Łukaszewicz [8]. Semen was di-
There is also an opinion that the poor fresh semen luted with EK diluent (1.4 g sodium glutamate, 0.14 g
quality would consequently give very poor frozen se- potassium citrate ⫻ H2O, 0.7 g glucose, 0.2 g D-fructose,
men characteristics [2]. 0.7 g inositol, 0.1 g polyvinylpyrrolidone, 0.02 g prota-
There are many methods of assessing semen quality mine sulfate, 0.98 g anhydrous sodium hydrogen phos-
and estimating the fertilising potential of spermatozoa. phate, 0.21 g anhydrous sodium dihydrogen phosphate
Some of them are regarded as subjective, others require were diluted to 100 mL with distilled water; pH 7.3,
special laboratory facilities. Traditional methods of se- osmotic pressure 390 mOsmol/kg) [9] at the ratio of 2:1.
men evaluation used to assess the quality of semen have After 15 min of equilibration at 4 °C the samples were
involved an estimation of the percentage of motile supplemented with dimethyloformamide (DMF) to a final
spermatozoa (on a pre-warmed glass slide), the sper- concentration of 6% and frozen to ⫺140 °C at a rate of
matozoa morphology (with various staining techni- 60 °C/min, in plastic straws (0.25 mL), in a programmable
ques), and the concentration in a unit dose (using freezing unit (Minidigitcoll 1400” IMV Technologies).
counting chamber). Conventional light microscopic se- Frozen samples were thawed in a water bath at 60 °C.
men assessment is being increasingly replaced by flu- The freezing procedure was performed ten times.
orescent staining techniques, computer-assisted sperm 2.4. Semen evaluation
analysis (CASA) system, and flow cytometry [3– 6].
To the best knowledge of the authors there are no The characteristics of the sperm were evaluated in
reports on cytometric evaluation of goose sperm char- the fresh and the frozen-thawed samples.
acteristics. Therefore, the present study was conducted The measurements were done on a FACSCalibur
to investigate spermatozoal viability, acrosome integ- (Becton Dickinson, San Jose, CA, USA) flow cytome-
rity, mitochondrial activity, and chromatin structure in ter. The fluorescent probes used in the experiment were
fresh and frozen-thawed Canada goose (Branta ca- excited by an Argon ion 488 nm laser.
nadensis) semen using flow cytometry. Acquisitions were done using the CellQuest 3.3 soft-
ware (Becton Dickinson). The non-sperm events were
gated out based on scatter properties and not analyzed.
2. Materials and methods A total of 40,000 events were analyzed for each sample.
2.1. Animals
2.4.1. Plasma membrane integrity
Ten 2-year-old Canada goose ganders (Branta ca- Sperm membrane integrity was assessed with dual
nadensis) were kept in individual cages (70 ⫻ 95 ⫻ 85 fluorescent probes SYBR-14 and propidium iodide (PI)
cm) under natural light and temperature conditions and (Live/Dead Sperm Viability Kit, InvitrogenTM, Eugene,
with access to a pool of water. Birds were fed with a OR, USA). The fresh and the frozen-thawed samples
dose of 250 –300g/day of commercial feed for breeding were diluted with EK diluent to a concentration of 50 ⫻
goose, containing 11.7 MJ metabolic energy and 140 g 106 spermatozoa per mL. Aliquots of 300 L sperm
crude protein per kg. suspension were mixed with 5 L of SYBR-14 work-A. Partyka et al. / Theriogenology 76 (2011) 843– 850 845
ing solution, and the mixture was incubated at room
temperature for 10 min. The working solution was
obtained by diluting a commercial solution of
SYBR-14 in distilled water at the ratio of 1:49. After
incubation the cells were counterstained with 5 L PI 5
min before analysis [3].
The four subpopulations of the events analyzed by
flow cytometer were noted by creating two dimensional
dot plots of PI (detector FL1) versus SYBR-14 (detector
FL2) fluorescence (Fig. 1). The PI⫺ SYBR- population
was regarded as debris and was not taken into account.
The percentage of sperm in the rest of the populations was
adjusted to 100%. The PI⫺ SYBR⫹ population was PI
negative, but stained by SYBR-14 and showed green
fluorescence, indicating that these cells had plasma mem-
brane intact. The PI⫹ SYBR- population contained cells
with red fluorescence and no sign of SYBR-14 fluores-
cence, which indicated that these cells were dead and the
PI⫹ SYBR⫹ population showing SYBR-14 and PI pos-
itive staining were considered to be dying. Fig. 2. Flow cytometric dot plot of Canada goose (Branta canadensis)
sperm analyzed for both PNA Alexa Fluor® and propidium iodide
2.4.2. Acrosome integrity (PI) fluorescence. PI⫺ PNA⫺ quadrant contains live cells with intact
Acrosomal damage was assessed using lectin PNA acrosome; PI⫺ PNA⫹ quadrant contains live cells with damaged
from Arachis hypogaea (peanut) Alexa Fluor® 488 acrosome; PI⫹ PNA⫺ quadrant contains dead cells with intact ac-
conjugate (InvitrogenTM, Eugene, OR, USA). PNA rosome; and PI⫹ PNA⫹ quadrant contains dead cells with damaged
working solution (10 L; 1 g/mL) was added to 500 acrosome.
L of diluted semen samples (50 ⫻ 106 spermatozoa
per mL) and incubated for 5 min in room temperature
in the dark. Following incubation, the supernatant was
removed by centrifugation (500 ⫻ g for 3 min) and the
sperm pellets were resuspended in 500 L of EK,
before cytometric analysis PI (5 L) was added to the
samples [3].
Dot plots of PNA/PI-stained spermatozoa showed
four populations of cells (Fig. 2). The quadrants were
set to determine and measure the percentages of the
following subpopulations: live cells with intact acro-
some (PI⫺ PNA⫺), live cells with ruptured acrosome
(PI⫺ PNA⫹), dead cells with intact acrosome (PI⫹
PNA⫺) and dead cells with ruptured acrosome (PI⫹
PNA⫹). Alexa Fluor® 488 signal was detected on
detector FL2 and PI fluorescence was detected on de-
tector FL1 on flow cytometer.
2.4.3. Mitochondrial function
The percentage of spermatozoa with functional mi-
Fig. 1. Flow cytometric dot plot of Canada goose (Branta canadensis) tochondria was estimated by combining fluorescent
sperm analyzed for both SYBR-14 and propidium iodide (PI) fluo- stains: Rhodamine 123 (R123; InvitrogenTM, Eugene,
rescence. PI⫺ SYBR- quadrant contains debris; PI⫺ SYBR⫹ quad-
rant contains live spermatozoa; PI⫹ SYBR- quadrant contains dead
OR, USA) and PI. R123 solution (10 L) was added to
spermatozoa; and PI⫹ SYBR⫹ quadrant contains dying spermato- 500 L of diluted semen samples (50 ⫻ 106 sperma-
zoa. tozoa per mL) and incubated for 20 min in room tem-846 A. Partyka et al. / Theriogenology 76 (2011) 843– 850
The sperm chromatin damage of spermatozoa was
quantified by the metachromatic shift from green (native,
double-stranded DNA) to red (denatured, single-stranded
DNA) fluorescence and displayed as red vs. green (Fig. 4).
Green florescence was detected on FL1 detector and red
fluorescence with detector FL3 on flow cytometer. The
main population represented the spermatozoa that emit
more green fluorescence than red fluorescence due to the
predominantly normal double-stranded configuration of
their DNA. Sperm cells located to the right of this main
population represented those cells which showed an in-
creased amount of red fluorescence and a decrease in
green fluorescence, compared with spermatozoa in the
main population. The following calculations were per-
formed for each sample: the percentage of spermatozoa
outside the main population with denatured DNA (%
DFI), the percentage of spermatozoa with an abnormally
high DNA stain ability–immature cells (% HDS). The
percentage of HDS cells was calculated by setting the
appropriate gate above the upper border of the main
Fig. 3. Flow cytometric dot plot of Canada goose (Branta canadensis) cluster of the sperm population with no detectable DNA
sperm analyzed for both Rhodamine 123 (Rh123) and propidium denaturation.
iodide (PI) fluorescence. PI⫺ R123⫺ quadrant contains live sperma-
tozoa with inactive mitochondria; PI⫺ R123⫹ quadrant contains live 2.5. Data analysis
spermatozoa with active mitochondria; PI⫹ R123-and R123⫹ quad-
rants contain dead spermatozoa. Statistical analyses were performed using STATISTICA
(StatSoft, Inc. (2001), version 6) [12]. The results ob-
perature in dark. Samples were then centrifuged at
500 ⫻ g for 3 min and the sperm pellets were resus-
pended in 500 L EK. Then 5 L of PI was added [10].
Populations of spermatozoa were identified accord-
ing to their green and red fluorescence after staining
with R123 (detector FL2) and PI (detector FL1), re-
spectively (Fig. 3). The quadrants were set to determine
and measure the percentage of the following subpo-
pulations: dead spermatozoa (PI⫹ R123⫺ and PI⫹
R123⫹), live spermatozoa with an inactive mitochon-
dria (PI⫺ R123⫺), and live spermatozoa with an active
mitochondria (PI⫺ R123⫹).
2.4.4. Assessment of chromatin status (SCSA)
Semen samples were diluted in EK diluent to a final
concentration of 1 ⫻ 106 spermatozoa per mL. The
suspension (200 L) was subjected to brief acid dena-
turation by mixing with 400 L of lysis solution (Triton
X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4)
held for 30 s and mixed with 1.2 mL acridine orange Fig. 4. Dot plot of the distribution of Canada goose (Branta canaden-
solution (AO; InvitrogenTM, Eugene, OR, USA) (6 g sis) spermatozoa based on green (FL1) and red (FL3) fluorescence.
Main population includes sperm without DNA fragmentation, %DFI
AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, represents the percentage of sperm with detectable DNA fragmenta-
EDTA 1 mM, NaCl 0.15 M, pH 6). After 3 min samples tion and % HDS determines the percentage of spermatozoa with an
were aspirated into the flow cytometer [11]. abnormally high DNA stain ability (immature cells).A. Partyka et al. / Theriogenology 76 (2011) 843– 850 847
Table 1 and freezing and thawing rates and temperatures
Plasma membrane integrity of Canada goose (Branta canadensis) [14,20]. In the present work we evidenced that the
spermatozoa in fresh and frozen-thawed semen (results expressed
as mean ⫾ SD).
cryopreservation of Canada goose semen significantly
decreased the percentage of live cells to 50% (P ⬍
Spermatozoa (%) Fresh semen Frozen-thawed semen
0.01) (Table 1). A similar result was obtained by Gee
Live (PI⫺ SYBR⫹) 76.3 ⫾ 9.6A 50.4 ⫾ 6.8B and Sexton [18] for Aleutian Brent goose using 6%
Dead (PI⫹ SYBR-) 20.3 ⫾ 6.2A 44.8 ⫾ 3.1B
Dying (PI⫹ SYBR⫹) 3.4 ⫾ 0.5 4.8 ⫾ 1.6
DMSO as the cryoprotectant. However, Tai et al
[19], using 4% DMSO and 9% DMA, received only
Different superscripts within lines indicate significant differences:
A,B
P ⬍ 0.01 (N ⫽ 10).
7.3% and 27% of live spermatozoa, respectively. Łu-
kaszewicz [8], using the same protocol of cryopreser-
vation as in our study, showed 68.4% of live sperma-
tained are presented as mean ⫾ SD of measurements on tozoa after thawing White Italian gander semen and
samples from 10 replicate determinations and were 62.1% of live cells in wild Greylag gander semen [15].
analyzed by ANOVA and Duncan’s multiple range test. Nevertheless, as we reported in the previous study [3],
All percentage data were transformed to arc sin prior to assessment of spermatozoa viability using SYBR-14
analyses. have demonstrated a lower percentage of live sperm in
the semen. Moreover, Gee and Sexton [18] reported
3. Results and discussion that they regarded the spermatozoa as live when 50% or
more of the particular cell area was unstained by eosin.
The flow cytometry commonly used in assessment This might explain the lower results of Canada goose
of mammal semen is progressively more often used for sperm viability in the fresh and frozen-thawed semen
determination of more detailed sperm characteristics in obtained in our experiment, as the authors mentioned
avian semen. In our previous studies [3,4], we showed above might also have included dying spermatozoa to
for the first time the accurate flow cytometric evalua- the live subpopulation.
tion of the fresh and frozen-thawed fowl sperm quality. The next feature that was assessed in our study was
The present study is also the first report describing the the acrosome integrity. In mammals the most com-
use of this technique for goose semen assessment. monly used method for the acrosome evaluation is the
The semen of birds intended for short-term storage plant lectin labeled by fluorescent probe [6,21–23]. In
or cryopreservation should maintain a very high quality the present study, we observed the significant decrease
[13]. The study of Łukaszewicz [14] showed that in the in the percentage of live spermatozoa with intact acro-
cryopreservation of goose sperm, particular attention some and also significant increase in percentage of dead
should be paid to the quality of the semen intended for cells with ruptured and intact acrosome after freezing-
freezing, since that factor largely influences post-thaw thawing procedure (P ⬍ 0.01) (Table 2). However, it is
gander sperm viability. The quantity and quality of noteworthy that after thawing, 50% of Canada goose
fresh semen depends on individual gander features, live spermatozoa retained intact acrosomes. Moreover,
as was reported in other species [5,15–17]. Table 1
showed plasma membrane integrity of spermatozoa in
the fresh and frozen-thawed semen. In our study the
Table 2
quality of the fresh Canada goose semen was not ex- The acrosome integrity of Canada goose (Branta canadensis)
cellent. We found 76.3% of live spermatozoa, which spermatozoa in fresh and frozen-thawed semen (results expressed
was lower than that obtained by Gee and Sexton [18], as mean ⫾ SD).
who reported 92.9% of live sperm cells in Aleutian Spermatozoa (%) Fresh semen Frozen-thawed
Canada goose (Branta canadensis leucopareia) on eo- semen
sin-nigrosin-stained slides. Other authors also showed a Live with intact acrosome 65.3 ⫾ 5.9A 50.4 ⫾ 5.0B
higher percentage of live spermatozoa: in White Italian (PI⫺ PNA⫺)
(Anser anser) gander semen, 92.2% [8]; in Chinese Live with ruptured acrosome 4.6 ⫾ 0.6 3.8 ⫾ 0.8
(PI⫺ PNA⫹)
Brown Geese, 83% [19]; and in Greylag ganders from
Dead with intact acrosome 28.1 ⫾ 5.9A 42.4 ⫾ 4.8B
90.3 to 93.3% [15]. (PI⫹ PNA⫺)
The efficacy of avian semen cryopreservation de- Dead with ruptured acrosome 2.0 ⫾ 0.6A 3.4 ⫾ 0.7B
pends on many factors, mainly those associated with (PI⫹ PNA⫹)
species, breed, the freezing medium (extenders and Different superscripts within lines indicate significant differences:
cryoprotectants), procedures of semen equilibration, A,B
P ⬍ 0.01 (N ⫽ 10).848 A. Partyka et al. / Theriogenology 76 (2011) 843– 850
the cryopreservation did not increase the percentage of Table 4
live spermatozoa with ruptured acrosome. That is con- Sperm chromatin structure assay results in fresh and frozen-thawed
semen of Canada goose (Branta canadensis); (results expressed as
sistent with our previous study [3], but there we found mean ⫾ SD).
that after freezing-thawing of fowl semen, only 18% of
Spermatozoa (%) Fresh Frozen-thawed
live spermatozoa had intact acrosome. Scanning elec-
tron microscopy studies conducted by Maeda et al DFI % 15.0 ⫾ 6.7 15.1 ⫾ 7.9
HDS % 5.4 ⫾ 1.8 6.9 ⫾ 3.2
[24,25] showed that acrosome damage of frozen-
thawed avian sperm are probably caused by the in- % DFI, the percentage of spermatozoa with DNA fragmentation; %
HDS, the percentage of spermatozoa with immature chromatin (less
crease in osmotic pressure, due to the concentration of chromatin condensation); (N ⫽ 10).
dissolved substances in diluents during cryopreserva-
tion. Therefore, it might be stated that the protocol used
in this study was appropriate for Canada goose semen thawing in Canada goose semen (Table 4). Similar
and could not cause an injury to sperm acrosomes. results were obtained Madeddu et al [29], who reported
Previous studies have shown that the midpiece ap- that chicken and Barbary partridge (Alecoris Barbara)
pears to be a sensitive component of avian sperm and spermatozoa were not particularly susceptible to DNA
the semen cryopreservation leads to the mitochondria fragmentation during cryopreservation, as assessed by
damage [3,26,27]. This is due to a loss of ATP, which comet assay. This is contrary to our previous experi-
supports multiple cellular activity and biochemical ment [3], in which we found that the cryopreservation
events, each required for successful fertilization of the fowl semen led to DNA defragmentation, and the
[28,29]. In the present study, we observed that the DFI in the fresh chicken semen was 1%, and rose after
percentage of live spermatozoa with mitochondrial ac- freezing-thawing up to 6%. However, the DFI results
tivity was significantly lower in the frozen-thawed se- we obtained in Canada geese semen evaluated by
men than in the fresh semen (P ⬍ 0.05) (Table 3). SCSA were notably higher and approached 15%. Fur-
However, almost 50% of live cells retained the active thermore, we could thus emphasize that even though
mitochondria. In the chicken semen the cryopreserva- the fresh goose semen had a poor quality, the cryo-
tion led to the decrease to 28% of live sperm with preservation of this semen did not provoke spermatozoa
mitochondrial activity [3], and also to the large decline DNA defragmentation. Moreover, there is evidence that
of ATP [29]. Therefore it could be stated, that species- other biochemical factors could play an important role
specific differences in spermatozoal ability to survive in sperm DNA stability during cryopreservation. Pre-
the cryopreservation process might be related to the vious studies have shown that human sperm DNA frag-
differences in the spermatozoa metabolic requirements. mentation was associated with an increase in oxidative
Moreover, the freezing protocol used in our experiment stress during freezing-thawing procedures and that the
was suitable to preserve 50% of the gamete of Canada addition of antioxidants to the cryoprotectant had a
goose which were able to withstand the freezing and significant protective effect on sperm DNA [30,31].
thawing process. Recently, we have reported that cryopreservation of
DNA integrity has also been considered as an im- White Koluda geese semen did not enhance lipid per-
portant parameter in the determination of spermatozoa oxidation in the live spermatozoa [4]. Therefore, we
ability to withstand the cryopreservation process. In the might speculate that the antioxidant defense in the
present study, we observed no changes in the percent- goose semen could be better than in the chicken semen
age of sperm with fragmented DNA after freezing- after thawing. Further studies should be conducted to
confirm this hypothesis.
In conclusion, so far, many cryopreservation proce-
Table 3
dures for domestic and wild avian species have been
The percentage of Canada goose (Branta canadensis) spermatozoa
with functional mitochondria in fresh and frozen-thawed semen developed. However, there are wide variations in the
(results expressed as mean ⫾ SD). results obtained, which are mainly affected by the cryo-
Spermatozoa (%) Fresh Frozen-thawed
protectant used, equilibration time, the quality of se-
men, sperm concentration, and volume of insemination
Live with active mitochondria 65.9 ⫾ 12.3 a
49.5 ⫾ 5.2b
(PI⫺/R123⫹) dose, duration, and frequency of insemination [32]. The
Live with inactive mitochondria 5.2 ⫾ 8.0 1.4 ⫾ 0.9 freezing of sperm induces the structural damage, and
(PI⫺/R123⫺) recently it has also been proved that this process affects
Different superscripts within lines indicate significant differences: the glycoproteins presence on the surface of sperm cells
P ⬍ 0.05 (N ⫽ 10).
a,b
[33], which are responsible for gamete recognition andA. Partyka et al. / Theriogenology 76 (2011) 843– 850 849
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