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 Niz˙an´skia 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

* Corresponding author. Tel.: ⫹48 71 32 05 300; fax: ⫹48 71 32 01 006. E-mail address: [email protected] (A. Partyka). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.04.016

In goose breeding, artificial insemination is not as commonly used as in turkeys or chickens. Compared to mammalian or chicken semen, the data on freezing gander semen are rather limited. This is partly caused

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by the low reproductive capability, resulting from the poor semen quality, low egg production, fertility, and hatchability rates that characterized geese in comparison to other poultry species. Moreover, geese have a relatively short reproductive period and ganders produce a small volume of ejaculate (0.05–1.0 mL) with a low spermatozoa concentration (0.03– 0.8 ⫻ 109/mL) and a low number of live normal cells (10 – 60%) [1]. However, the possibility of semen storage in the liquid nitrogen for an unlimited period may be helpful in wild goose gene pool preservation. It is well acknowledged, in practice, that one of the criteria to predict the freezability of spermatozoa is the quality of the fresh semen. Cryopreservation induces many unfavorable changes in spermatozoa that may lead to cell injury and cause lower quality of frozenthawed semen in comparison with a fresh ejaculate. There is also an opinion that the poor fresh semen quality would consequently give very poor frozen semen characteristics [2]. There are many methods of assessing semen quality and estimating the fertilising potential of spermatozoa. Some of them are regarded as subjective, others require special laboratory facilities. Traditional methods of semen evaluation used to assess the quality of semen have involved an estimation of the percentage of motile spermatozoa (on a pre-warmed glass slide), the spermatozoa morphology (with various staining techniques), and the concentration in a unit dose (using counting chamber). Conventional light microscopic semen assessment is being increasingly replaced by fluorescent staining techniques, computer-assisted sperm analysis (CASA) system, and flow cytometry [3– 6]. To the best knowledge of the authors there are no reports on cytometric evaluation of goose sperm characteristics. Therefore, the present study was conducted to investigate spermatozoal viability, acrosome integrity, mitochondrial activity, and chromatin structure in fresh and frozen-thawed Canada goose (Branta canadensis) semen using flow cytometry. 2. Materials and methods 2.1. Animals Ten 2-year-old Canada goose ganders (Branta canadensis) were kept in individual cages (70 ⫻ 95 ⫻ 85 cm) under natural light and temperature conditions and with access to a pool of water. Birds were fed with a dose of 250 –300g/day of commercial feed for breeding goose, containing 11.7 MJ metabolic energy and 140 g crude protein per kg.

2.2. Semen collection procedure Semen of ten males was collected two times a week by the dorso-abdominal massage method [7] and then pooled to obtain a sufficient sample for analysis. During the semen collection particular care was taken to minimize the contamination of semen with uric acid or feces. After collection the pool was split into two aliquots, one for fresh semen evaluation and the other one for the cryopreservation procedure. During one reproductive cycle, 10 semen collections, from each male, were performed. 2.3. Semen cryopreservation Within 20 min following the collection, semen samples were subjected to cryopreservation in accordance with the procedure of Łukaszewicz [8]. Semen was diluted with EK diluent (1.4 g sodium glutamate, 0.14 g potassium citrate ⫻ H2O, 0.7 g glucose, 0.2 g D-fructose, 0.7 g inositol, 0.1 g polyvinylpyrrolidone, 0.02 g protamine sulfate, 0.98 g anhydrous sodium hydrogen phosphate, 0.21 g anhydrous sodium dihydrogen phosphate were diluted to 100 mL with distilled water; pH 7.3, osmotic pressure 390 mOsmol/kg) [9] at the ratio of 2:1. After 15 min of equilibration at 4 °C the samples were supplemented with dimethyloformamide (DMF) to a final concentration of 6% and frozen to ⫺140 °C at a rate of 60 °C/min, in plastic straws (0.25 mL), in a programmable freezing unit (Minidigitcoll 1400” IMV Technologies). Frozen samples were thawed in a water bath at 60 °C. The freezing procedure was performed ten times. 2.4. Semen evaluation The characteristics of the sperm were evaluated in the fresh and the frozen-thawed samples. The measurements were done on a FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. The fluorescent probes used in the experiment were excited by an Argon ion 488 nm laser. Acquisitions were done using the CellQuest 3.3 software (Becton Dickinson). The non-sperm events were gated out based on scatter properties and not analyzed. A total of 40,000 events were analyzed for each sample. 2.4.1. Plasma membrane integrity Sperm membrane integrity was assessed with dual fluorescent probes SYBR-14 and propidium iodide (PI) (Live/Dead Sperm Viability Kit, InvitrogenTM, Eugene, OR, USA). The fresh and the frozen-thawed samples were diluted with EK diluent to a concentration of 50 ⫻ 106 spermatozoa per mL. Aliquots of 300 ␮L sperm suspension were mixed with 5 ␮L of SYBR-14 work-

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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 membrane intact. The PI⫹ SYBR- population contained cells with red fluorescence and no sign of SYBR-14 fluorescence, which indicated that these cells were dead and the PI⫹ SYBR⫹ population showing SYBR-14 and PI positive staining were considered to be dying. 2.4.2. Acrosome integrity Acrosomal damage was assessed using lectin PNA from Arachis hypogaea (peanut) Alexa Fluor® 488 conjugate (InvitrogenTM, Eugene, OR, USA). PNA working solution (10 ␮L; 1 ␮g/mL) was added to 500

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Fig. 2. Flow cytometric dot plot of Canada goose (Branta canadensis) sperm analyzed for both PNA Alexa Fluor® and propidium iodide (PI) fluorescence. PI⫺ PNA⫺ quadrant contains live cells with intact acrosome; PI⫺ PNA⫹ quadrant contains live cells with damaged acrosome; PI⫹ PNA⫺ quadrant contains dead cells with intact acrosome; and PI⫹ PNA⫹ quadrant contains dead cells with damaged 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 acrosome (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 detector FL1 on flow cytometer.

Fig. 1. Flow cytometric dot plot of Canada goose (Branta canadensis) sperm analyzed for both SYBR-14 and propidium iodide (PI) fluorescence. PI⫺ SYBR- quadrant contains debris; PI⫺ SYBR⫹ quadrant contains live spermatozoa; PI⫹ SYBR- quadrant contains dead spermatozoa; and PI⫹ SYBR⫹ quadrant contains dying spermatozoa.

2.4.3. Mitochondrial function The percentage of spermatozoa with functional mitochondria was estimated by combining fluorescent stains: Rhodamine 123 (R123; InvitrogenTM, Eugene, OR, USA) and PI. R123 solution (10 ␮L) was added to 500 ␮L of diluted semen samples (50 ⫻ 106 spermatozoa per mL) and incubated for 20 min in room tem-

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Fig. 3. Flow cytometric dot plot of Canada goose (Branta canadensis) sperm analyzed for both Rhodamine 123 (Rh123) and propidium iodide (PI) fluorescence. PI⫺ R123⫺ quadrant contains live spermatozoa with inactive mitochondria; PI⫺ R123⫹ quadrant contains live spermatozoa with active mitochondria; PI⫹ R123-and R123⫹ quadrants contain dead spermatozoa.

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 increased amount of red fluorescence and a decrease in green fluorescence, compared with spermatozoa in the main population. The following calculations were performed 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 cluster of the sperm population with no detectable DNA denaturation. 2.5. Data analysis 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 resuspended in 500 ␮L EK. Then 5 ␮L of PI was added [10]. Populations of spermatozoa were identified according to their green and red fluorescence after staining with R123 (detector FL2) and PI (detector FL1), respectively (Fig. 3). The quadrants were set to determine and measure the percentage of the following subpopulations: dead spermatozoa (PI⫹ R123⫺ and PI⫹ R123⫹), live spermatozoa with an inactive mitochondria (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 denaturation 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 solution (AO; InvitrogenTM, Eugene, OR, USA) (6 ␮g AO/mL in a buffer: citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl 0.15 M, pH 6). After 3 min samples were aspirated into the flow cytometer [11].

Fig. 4. Dot plot of the distribution of Canada goose (Branta canadensis) spermatozoa based on green (FL1) and red (FL3) fluorescence. Main population includes sperm without DNA fragmentation, %DFI represents the percentage of sperm with detectable DNA fragmentation and % HDS determines the percentage of spermatozoa with an abnormally high DNA stain ability (immature cells).

A. Partyka et al. / Theriogenology 76 (2011) 843– 850 Table 1 Plasma membrane integrity of Canada goose (Branta canadensis) spermatozoa in fresh and frozen-thawed semen (results expressed as mean ⫾ SD). Spermatozoa (%)

Fresh semen

Frozen-thawed semen

Live (PI⫺ SYBR⫹) Dead (PI⫹ SYBR-) Dying (PI⫹ SYBR⫹)

76.3 ⫾ 9.6A 20.3 ⫾ 6.2A 3.4 ⫾ 0.5

50.4 ⫾ 6.8B 44.8 ⫾ 3.1B 4.8 ⫾ 1.6

Different superscripts within lines indicate significant differences: A,B P ⬍ 0.01 (N ⫽ 10).

tained are presented as mean ⫾ SD of measurements on samples from 10 replicate determinations and were analyzed by ANOVA and Duncan’s multiple range test. All percentage data were transformed to arc sin prior to analyses. 3. Results and discussion The flow cytometry commonly used in assessment of mammal semen is progressively more often used for determination of more detailed sperm characteristics in avian semen. In our previous studies [3,4], we showed for the first time the accurate flow cytometric evaluation of the fresh and frozen-thawed fowl sperm quality. The present study is also the first report describing the use of this technique for goose semen assessment. The semen of birds intended for short-term storage or cryopreservation should maintain a very high quality [13]. The study of Łukaszewicz [14] showed that in the cryopreservation of goose sperm, particular attention should be paid to the quality of the semen intended for freezing, since that factor largely influences post-thaw gander sperm viability. The quantity and quality of fresh semen depends on individual gander features, 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 quality of the fresh Canada goose semen was not excellent. We found 76.3% of live spermatozoa, which was lower than that obtained by Gee and Sexton [18], who reported 92.9% of live sperm cells in Aleutian Canada goose (Branta canadensis leucopareia) on eosin-nigrosin-stained slides. Other authors also showed a higher percentage of live spermatozoa: in White Italian (Anser anser) gander semen, 92.2% [8]; in Chinese Brown Geese, 83% [19]; and in Greylag ganders from 90.3 to 93.3% [15]. The efficacy of avian semen cryopreservation depends on many factors, mainly those associated with species, breed, the freezing medium (extenders and cryoprotectants), procedures of semen equilibration,

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and freezing and thawing rates and temperatures [14,20]. In the present work we evidenced that the cryopreservation of Canada goose semen significantly decreased the percentage of live cells to 50% (P ⬍ 0.01) (Table 1). A similar result was obtained by Gee and Sexton [18] for Aleutian Brent goose using 6% DMSO as the cryoprotectant. However, Tai et al [19], using 4% DMSO and 9% DMA, received only 7.3% and 27% of live spermatozoa, respectively. Łukaszewicz [8], using the same protocol of cryopreservation as in our study, showed 68.4% of live spermatozoa after thawing White Italian gander semen and 62.1% of live cells in wild Greylag gander semen [15]. Nevertheless, as we reported in the previous study [3], assessment of spermatozoa viability using SYBR-14 have demonstrated a lower percentage of live sperm in the semen. Moreover, Gee and Sexton [18] reported that they regarded the spermatozoa as live when 50% or more of the particular cell area was unstained by eosin. This might explain the lower results of Canada goose sperm viability in the fresh and frozen-thawed semen obtained in our experiment, as the authors mentioned above might also have included dying spermatozoa to the live subpopulation. The next feature that was assessed in our study was the acrosome integrity. In mammals the most commonly used method for the acrosome evaluation is the plant lectin labeled by fluorescent probe [6,21–23]. In the present study, we observed the significant decrease in the percentage of live spermatozoa with intact acrosome and also significant increase in percentage of dead cells with ruptured and intact acrosome after freezingthawing procedure (P ⬍ 0.01) (Table 2). However, it is noteworthy that after thawing, 50% of Canada goose live spermatozoa retained intact acrosomes. Moreover,

Table 2 The acrosome integrity of Canada goose (Branta canadensis) spermatozoa in fresh and frozen-thawed semen (results expressed as mean ⫾ SD). Spermatozoa (%)

Fresh semen

Frozen-thawed semen

Live with intact acrosome (PI⫺ PNA⫺) Live with ruptured acrosome (PI⫺ PNA⫹) Dead with intact acrosome (PI⫹ PNA⫺) Dead with ruptured acrosome (PI⫹ PNA⫹)

65.3 ⫾ 5.9A

50.4 ⫾ 5.0B

4.6 ⫾ 0.6

3.8 ⫾ 0.8

28.1 ⫾ 5.9A

42.4 ⫾ 4.8B

2.0 ⫾ 0.6A

3.4 ⫾ 0.7B

Different superscripts within lines indicate significant differences: A,B P ⬍ 0.01 (N ⫽ 10).

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the cryopreservation did not increase the percentage of live spermatozoa with ruptured acrosome. That is consistent with our previous study [3], but there we found that after freezing-thawing of fowl semen, only 18% of live spermatozoa had intact acrosome. Scanning electron microscopy studies conducted by Maeda et al [24,25] showed that acrosome damage of frozenthawed avian sperm are probably caused by the increase in osmotic pressure, due to the concentration of dissolved substances in diluents during cryopreservation. Therefore, it might be stated that the protocol used in this study was appropriate for Canada goose semen and could not cause an injury to sperm acrosomes. Previous studies have shown that the midpiece appears to be a sensitive component of avian sperm and the semen cryopreservation leads to the mitochondria damage [3,26,27]. This is due to a loss of ATP, which supports multiple cellular activity and biochemical events, each required for successful fertilization [28,29]. In the present study, we observed that the percentage of live spermatozoa with mitochondrial activity was significantly lower in the frozen-thawed semen than in the fresh semen (P ⬍ 0.05) (Table 3). However, almost 50% of live cells retained the active mitochondria. In the chicken semen the cryopreservation led to the decrease to 28% of live sperm with mitochondrial activity [3], and also to the large decline of ATP [29]. Therefore it could be stated, that speciesspecific differences in spermatozoal ability to survive the cryopreservation process might be related to the differences in the spermatozoa metabolic requirements. Moreover, the freezing protocol used in our experiment was suitable to preserve 50% of the gamete of Canada goose which were able to withstand the freezing and thawing process. DNA integrity has also been considered as an important parameter in the determination of spermatozoa ability to withstand the cryopreservation process. In the present study, we observed no changes in the percentage of sperm with fragmented DNA after freezingTable 3 The percentage of Canada goose (Branta canadensis) spermatozoa with functional mitochondria in fresh and frozen-thawed semen (results expressed as mean ⫾ SD). Spermatozoa (%) Live with active mitochondria (PI⫺/R123⫹) Live with inactive mitochondria (PI⫺/R123⫺)

Fresh

Frozen-thawed

65.9 ⫾ 12.3

49.5 ⫾ 5.2b

5.2 ⫾ 8.0

1.4 ⫾ 0.9

a

Different superscripts within lines indicate significant differences: P ⬍ 0.05 (N ⫽ 10).

a,b

Table 4 Sperm chromatin structure assay results in fresh and frozen-thawed semen of Canada goose (Branta canadensis); (results expressed as mean ⫾ SD). Spermatozoa (%) DFI % HDS %

Fresh

Frozen-thawed

15.0 ⫾ 6.7 5.4 ⫾ 1.8

15.1 ⫾ 7.9 6.9 ⫾ 3.2

% DFI, the percentage of spermatozoa with DNA fragmentation; % HDS, the percentage of spermatozoa with immature chromatin (less chromatin condensation); (N ⫽ 10).

thawing in Canada goose semen (Table 4). Similar results were obtained Madeddu et al [29], who reported that chicken and Barbary partridge (Alecoris Barbara) spermatozoa were not particularly susceptible to DNA fragmentation during cryopreservation, as assessed by comet assay. This is contrary to our previous experiment [3], in which we found that the cryopreservation of the fowl semen led to DNA defragmentation, and the DFI in the fresh chicken semen was 1%, and rose after freezing-thawing up to 6%. However, the DFI results we obtained in Canada geese semen evaluated by SCSA were notably higher and approached 15%. Furthermore, we could thus emphasize that even though the fresh goose semen had a poor quality, the cryopreservation of this semen did not provoke spermatozoa DNA defragmentation. Moreover, there is evidence that other biochemical factors could play an important role in sperm DNA stability during cryopreservation. Previous studies have shown that human sperm DNA fragmentation was associated with an increase in oxidative stress during freezing-thawing procedures and that the addition of antioxidants to the cryoprotectant had a significant protective effect on sperm DNA [30,31]. Recently, we have reported that cryopreservation of White Koluda geese semen did not enhance lipid peroxidation in the live spermatozoa [4]. Therefore, we might speculate that the antioxidant defense in the goose semen could be better than in the chicken semen after thawing. Further studies should be conducted to confirm this hypothesis. In conclusion, so far, many cryopreservation procedures for domestic and wild avian species have been developed. However, there are wide variations in the results obtained, which are mainly affected by the cryoprotectant used, equilibration time, the quality of semen, sperm concentration, and volume of insemination dose, duration, and frequency of insemination [32]. The freezing of sperm induces the structural damage, and recently it has also been proved that this process affects the glycoproteins presence on the surface of sperm cells [33], which are responsible for gamete recognition and

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the connection with the egg cell. However, Łukaszewicz [8] found no influence of the process of freezing White Koluda® goose semen on the effectiveness of sperm fertilizing ability. Our study showed that, even though the fresh Canada goose semen was of poor quality, the cryopreservation did not provoke spermatozoa DNA defragmentation and injury of acrosomes, and almost 50% of spermatozoa withstood the freezingthawing process and retained the mitochondrial activity.

Acknowledgments This study was supported by the Polish Ministry of Science and Higher Education, grant no. N N311 2217 33. The authors wish to thank Dr Maria Chrzanowska for technical support.

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