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Journal of Fish Biology (2016)
doi:10.1111/jfb.13245, available online at wileyonlinelibrary.com
Effects of storage time on the motility, mortality and
calcium levels of Atlantic salmon Salmo salar spermatozoa
J. P*†, G. G*, M. C‡,
A. R-R§ F. R‡
*Laboratorio de Biología Celular Aplicada, Núcleo de Investigación en Producción
Alimentaria, Escuela de medicina veterinaria, Facultad de recursos naturales, Universidad
Católica of Temuco, Chile, ‡Center of Neurosciences and Peptide Biology, Faculty of
Medicine, Universidad de La Frontera, Temuco, Chile and §Cryobiology and Spermatozoa
Functionality Analysis Laboratory, Institute of Animal Sciences, Faculty of Veterinary
Sciences, Universidad Austral de Chile, Valdivia, Chile
(Received 5 June 2015, Accepted 14 November 2016)
This study estimates spermatozoa mortality, morphology, motility and intracellular calcium levels in
Atlantic salmon Salmo salar milt after prolonged storage. Milt samples were preserved at 4∘Cfor
25 days and then evaluated for mortality. Motility remained high for the rst 3 days and the mortality
was low during the rst 5 days of storage.
A decrease of >50% in calcium content was observed after 5 days of storage. When spermatozoa were
activated, calcium levels increased >200% in relative uorescence units (RFU); this rate of increase
was lost when the samples were stored for extended periods of time and was only partially manifested
in a zero calcium solution. The results suggest that in vitro storage of S. salar spermatozoa at 4∘Cfora
period of 3 days preserves motility and limits mortality to levels similar to those of fresh spermatozoa.
This method also maintains intracellular calcium storage critical for spermatozoa performance.
© 2016 The Fisheries Society of the British Isles
Key words: conservation; function; milt; viability.
INTRODUCTION
In sh aquaculture, milt is commonly stored outside of the natural testicular envi-
ronment. This occurs, for example, when diagnostic examinations are required, or
if breeding is to occur at a different location (Cabrita et al., 2001; Martinez-Paramo
et al., 2009). One way to avoid the deterioration of stored spermatozoa is to dilute
the milt in either an isotonic saline solution (articial seminal uid), or in solutions
that mimic the testicular environment. It has been proposed that spermatozoa should
be preserved directly in milt (Bozkurt et al., 2005; Dziewulska et al., 2010). Studies
on spermatozoa quality in shes when stored in vitro usually include an evaluation
of characteristics such as colour, consistency, spermatozoa density, motility (Rawlings
et al., 1994; Crabtree, 2010; Hegedusova et al., 2010), or seminal plasma composi-
tion (Lahnsteiner et al., 1996). Currently, there is insufcient information regarding
†Author to whom correspondence should be addressed. Tel.: +56 45 2205564; email: jparodi@uct.cl
1
© 2016 The Fisheries Society of the British Isles
2J. PARODI ET AL.
decreased cell function and mortality in relation to intracellular calcium levels. Calcium
is a secondary cellular messenger that is required for complex functions in diverse
types of cells, including the regulation of gene expression, muscle contraction and
molecular transport, as well as the amplication of the action of ligands on the cell
surface (Zamburlin et al., 2012; Schwarz et al., 2013). The calcium ion is regulated
through a number of highly complex mechanisms, including pumps, ion channels and
intracellular reservoirs. Spermatozoa utilize many of these mechanisms to regulate
intracellular calcium concentration, including specic and highly regulated channels
such as cationic sperm channels (CatSper), voltage-dependent channels and intracellu-
lar mitochondrial calcium stores (Darszon et al., 2007). Such channels have not been
detected in sh spermatozoa, however, nor are the acrosome structures present. These
cells contain diverse conductance levels, which are part of the mechanism that regulates
hyperpolarization in mammalian spermatozoa cells (Darszon et al., 1999; Kho et al.,
2001). Nonetheless, motility is suppressed in salmonids by millimolar concentrations
of extracellular potassium in the milt. When there is a decrease in extracellular potas-
sium, such as in fresh water, it triggers signalling for the initiation of sperm motility
(Morisawa & Suzuki, 1980). This decrease in extracellular potassium can induce an
efux of ions through specic types of channels on the spermatozoa, which leads to a
hyperpolarization state of the plasma membrane (Tanimoto et al., 1994). Intracellular
calcium concentration subsequently increases. This change in calcium levels allows
an increase in spermatozoa motility, which lasts for a few minutes and then declines
(Cosson et al., 1989; Alavi & Cosson, 2006). These experimental data, however, do
not fully clarify the effects of different calcium levels on the activation of spermatozoa
motility, spermatozoa regulation of the entry and exit of calcium, or storage time and
its effect on basal calcium levels and motility activation (Gallego et al., 2013).
Various reports on salmonid spermatozoa have shown that calcium entry is regulated
by osmotic change, regardless of potassium changes. Various reports also demonstrate
the importance of intracellular calcium motility (Oda & Morisawa, 1993; Takei et al.,
2012). This study measured calcium levels in Atlantic salmon Salmo salar L. 1758
spermatozoa, preserved under laboratory conditions at 4∘C and shaken and aerated
every day in a commercial dilution solution, in order to nd the relationship between
the period of conservation and several physiological functions: spermatozoa mortality,
motility and kinetic and calcium levels.
MATERIALS AND METHODS
MILT SAMPLING
Milt was extracted from 20 mature 2 year-old male S. salar. The animals and biological
samples were handled in accordance with the manual guidelines of Universidad Católica of
Temuco (UCT) and CONICYT (Comisión Nacional de Investigación Cientíca y Tecnológica
de Chile) and approved by FONDEF (Fondo de Fomento al Desarrollo Cientíco y Tecnológico)
project D10i1064. All biological materials were discarded following the UCT protocol. Before
milt collection, sh were anaesthetized with 0·015% benzocaine (BZ-20; Veterquimica, www
.veterquimica.cl). The single milt sample from each male was then collected using abdominal
massages or stripping.
BIOACUI (Unidad de Biotecnologia Acuicola) donated a 1ml sample from every one of
the males used in the present study. The samples were subsequently oxygenated in a horizon-
tal shaker; each day fresh atmospheric air was pumped to the sample (Regent Calm RC-001;
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
ROLE OF CALCIUM ON S. SALAR SPERMATOZOA 3
Calm-KW, DongChen, China) inside the incubator with a single tubing to aerate the sample.
Stopmilt (BIOACUI; Temuco, Chile) was used to dilute the samples in a keeper solution (Ubilla,
2011), which are stored individually in sealed plastic containers, modied from a published
report (Lahnsteiner & Mansour, 2012). In the laboratory, milt was diluted at a ratio of 1:2 (5 ml of
milt: 10 ml of spermatozoa dilutant, Stopmilt, BIOACUI commercial solution). All procedures
were performed at 4∘C (Ubilla & Valdebenito, 2011).
MEASURING MORTALITY
To determine the mortality of S. salar spermatozoa cells, an eosin staining protocol was
followed. To measurement the mortality, a modication of the Olivares et al. (2015) mam-
malian protocol was used. Spermatozoa were maintained under laboratory conditions in an
incubator in order to maintain the sample temperature at 4∘C. Orbital rotators (compact digital
microplates, Thermo scientic; www.thermo-scientic.com) were placed inside the incubator
(BOD Incubator 2005, VWR; www.vwr.com) in order to shake the sample. One hundred μlwas
collected from the diluted milt sample on different days to measure mortality. An initial value,
taken on the rst day of the measurement of the sample was used as a control. The spermato-
zoa were maintained for 10 days and then evaluated. To generate a model of ageing, samples
were stored for 20 days, these samples were dened as aged sperm. Spermatozoa were kept
at 4∘C, incubated with 0·3% eosin in glacial acetic acid and distilled water for 1 min, then
removed from the solution and dried for observation. One hundred random spermatozoa cells
were counted and those with a red-orange colour were classied as dead. By modifying the
protocol used for mammalian cells, it was established as the protocol for the control of the
spermatozoa mortality rate. Phosphate buffer (PBS) and high temperatures are used to gener-
ate cell death. This strategy has a positive result, with a red colouration appearing when the
spermatozoa died.
MOTILITY AND KINETIC ASSESSMENT
A10μlsampleofS. salar 0 day-old spermatozoa was taken as a control each day and
stored for 10 days, every day a sub-sample was prepared on a slide and kept on ice to preserve
the temperature and then rapidly mounted on a Nikon Snapshot phase-contrast microscope
(www.nikon.com). Once the sample was in focus, 20 μl of commercial activation solution was
applied (Powermilt, BIOACUI). To observe the effect of calcium, the Powermilt activation
solution CaCl2 was replaced with KCl (5 g l−1). The same experiment was performed with
a solution containing EGTA [ethylene glycol-bis(𝛽-aminoethyl ether)-N,N,N′,N′-tetraacetic
acid; 0·4gl
−1] and then again with a solution without calcium (zero calcium). Both solutions
(normal and zero calcium) contained values of 260 mOsmol kg−1in order to induce osmotic
shock and spermatozoa activation.
All procedures were performed at 4∘C and are applied to each sample to activate sperma-
tozoa movement in the presence or absence of extracellular calcium. Powermilt solution was
used instead of coverslips in order to maintain access to the sample (Figueroa et al., 2013).
A time-lapse video was recorded from the initiation of cell movement until all cells within the
visual eld had stopped moving and kinetics were no longer observable by the image analysis
programme. Kinetics were measured using a low-cost computer-assisted sperm-analyser system
(CASA; ImageJ plug-in; https://imagej.nih.gov/) (Parodi et al., 2015). Video was recorded at
×10 with inverted illuminated-eld microscope and began immediately after the activation of
the spermatozoa cells.
INTRACELLULAR CALCIUM MEASUREMENTS
Spermatozoa cells were incubated in a diluent solution containing the uorescent probe,
Fluo-4 AM, at a working concentration of 0·5μMl
−1(1 mM stock in pluronic acid with
DMSO, Molecular Probes; ThermoFisher). All solutions and samples were maintained at
4∘C for 30 min to allow entry of the spermatozoa probe. The preparation was then washed
twice with the dilutant and incubated for 30 min. This protocol is a modied version of the
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
4J. PARODI ET AL.
method used for bovine spermatozoa cells (Navarrete et al., 2010). The preparations were
mounted on slides with coverslips and observed using a laser-scanning confocal microscope
coupled with a spectra detector (Olympus; www.olympus.com). The spermatozoa cells were
located using phase contrast and observed under uorescence; the images were obtained under
excitation at 480 nm. The results were expressed in relative uorescence units (RFU) as a ratio
of the baseline eld observed with the variation over time and plotted using the programme
ORIGIN 8.0, modied from Navarrete et al. (2012). Microuorimetry was used in order to
quickly observe changes in the calcium spike. The samples were loaded with the calcium
probe Fluo4 (Life Sciences; www.lifesciencesusa.com), transferred to a quartz cuvette and
then measured in a micro uorometer coupled with a photodetector. Samples were kept in a
circulating system at 4∘C and exposed to Powermilt in the presence and absence of extracellular
calcium (zero calcium solution). A recovery solution containing calcium was applied. Changes
were registered using FeliX-32 software (Photon Technology International Inc.; www.pti- nj
.com) with excitation at 488 nm and the samples were lit for a short time (time <0·266 s) in
each measured sample. Data were analysed with Origin 8.0 software and plotted as RFUs.
Spermatozoa cell autouorescence was not signicant and was used as the baseline for different
experiments.
IMAGE ANALYSIS
For immunouorescence observation and calcium quantitation, ImageJ was used to measure
the average intensity of the regions of interest (ROI) and the uorescence intensity proles of
spermatozoa cells loaded with uo-4 uorescent probes under different conditions: storage time
(from 0 to 10 days of conservation), active spermatozoa (in the presence of Powermilt) and active
ageing spermatozoa (at 20 days of conservation, using Powermilt). To obtain intensity proles,
a straight line was drawn through the cell, which the software automatically interpreted as a
graph, presenting the intensity in arbitrary units (0–255) to convey the average intensity of the
specied zone, which roughly corresponded to the contour of the cell. The average intensity of
the ROIs was calculated by drawing lines through the ROI of each cell studied.
STATISTICAL ANALYSIS
All results, including image analysis, unless indicated otherwise, are presented as the
mean ±.. ANOVA was applied to compare the effects of the number of days in storage.
Post-test Bonferroni analysis was applied in order to compare means with P<0·05. Similarly,
ANOVA or a t-test was considered statistically signicant at P<0·05. All data were analysed
using the Prism 4.0 statistical program (www.graphpad.com).
RESULTS
SALMO SALAR MILT AND PHYSIOLOGICAL VARIABLES
Salmo salar milt was stored for 10 days at 4∘C under control conditions. At day 0,
milt mortality was assessed [Fig. 1(a)]. The samples are expressed in percentages living
and dead cells at day 0 and at 10 days of being stored in the laboratory [Fig. 1(b)]. To
be usable, stored spermatozoa samples must exhibit normal physiological functions.
By examining the samples over time, it was seen that milt motility was reduced by
>50% after 5 days [Fig. 1(c)]. These results indicate that the cell viability can be mea-
sured and that the spermatozoa are viable and motile shortly after arrival of the sample
to the laboratory, as fresh ejaculate. Furthermore, the ndings show that methodolo-
gies widely used to study mammalian spermatozoa can be adapted for sample sh
spermatozoa.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
ROLE OF CALCIUM ON S. SALAR SPERMATOZOA 5
Control 3 days 20 days
2 µm
(b)
(a)
120
80
40
Live cells normalized (%)
0
–20246810 12
(c)
Days
120
80
40
Total motility normalized (%)
0
–2 0 2 4 6 810 12
(d) 200
150
50
VCL (µm s–1)
0
Control Activated
100
(e) 80
60
20
VSL (µm s–1)
0
Control Activated
40
*
**
*
*
*
*
*
*
*
*
*
*
*
F. 1. Effects of storage time on Salmo salar spermatozoa. (a) Microphotograph of spermatozoa stained with the
supravital technique after 0 (control), 3 and 20 days. (b) Time series of spermatozoa survival (mean ±..,
n=6) under storage conditions. *, P<0·05, ANOVA comparison with day 0. (c) Time series of changes in
S. salar spermatozoa motility. *, P<0·05, ANOVA comparison with day 0. (d) Mean ±.. (n=6) velocity
curvilinear line (VCL) rate over Powermilt. The control insert shows a non-motile sample; the insert within
the activated results shows the recorded traces of motile spermatozoa. *, P<0·05, ANOVA comparison with
day 0. (e) Mean ±.. (n=6) velocity straight-line (VCL) rate over Powermilt. The control insert shows a
non-motile sample; the insert within the activated results shows the recorded traces of motile spermatozoa.
*, P<0·05, ANOVA comparison with day 0.
PHYSIOLOGICAL VARIABLES OF MILT ON MAINTAINING
CONDITION
The samples were stable and viable in Stopmilt for up to 4 days. Mortality increased
after 4 days; a change was observed in diluent solution at day 5 of storage [Fig. 1(b)].
Motility was taken as a functional variable and could be induced by the use of Pow-
ermilt [Fig. 1(c)]. It was observed that the ability of spermatozoa to respond to the
activation stimulus declined with the storage time when compared with the initial
measurement of spermatozoa kept in diluent solution, although there were no signi-
cant changes until day 5 of storage. The velocity curvilinear line (VCL) and velocity
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
6J. PARODI ET AL.
Day 0
Day 20
5 µm
–2 0 2 4 6 810 12
0
40
80
120
Days
*
****
Calcium RFU
normalized (%)
(a) (b)
F. 2. Effects of storage time on calcium levels in Salmo salar spermatozoa. (a) Microphotograph of stained
spermatozoa with a calcium probe at day 0 and after 20 days. (b) Time series of mean ±.. (n=6) intra-
cellular calcium level, expressed as relative reference units (RFU), in spermatozoa after different lengths of
storage time. *, P<0·05, ANOVA comparison with day 0.
straight-line (VSL) kinetic variables were measured upon activating the sample after 5
days of storage [Fig. 1(d), (e), respectively]. The insets of these in the bar graph show
capturing of the spermatozoa movement, which was traced using a protocol-based sys-
tem including in ImageJ CASA software. The tracing helped to obtain kinetic values
(Parodi et al., 2015). When the samples were activated, spermatozoa responded and
showed measurable kinetic activity. These data suggest that spermatozoa present in
milt preserved for a short time under controlled conditions maintain a signicant value
of the tested variables, with few examples of mortality and motility of over 50%.
CHANGE IN INTRACELLULAR CALCIUM OF S. SALAR
SPERMATOZOA OVER TIME
Salmo salar spermatozoa cells were loaded with a uorescent probe to monitor
the calcium content. The loaded samples were activated for control and motility was
then observed. Photomicrographs were obtained using a confocal microscopy system
over the course of several days and the signal intensity of the ROI was quantied.
Micrographs of fresh samples (day 0) and 20 day-old spermatozoa (ageing) loaded
with the probes showed that no morphological change was observing when sper-
matozoa were loaded [Fig. 2(a)]. Daily uorescent measurements demonstrated that
although calcium content remains stable for 5 days, it declined signicantly after
6 days [Fig. 2(b)]. These data suggest that stable calcium content could be responsible
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
ROLE OF CALCIUM ON S. SALAR SPERMATOZOA 7
for maintaining the physiological function of spermatozoa and that prolonged periods
of storage results in calcium loss.
CHANGES IN CALCIUM AFFECT THE ACUTE PROCESSES
OF SPERMATOZOA
Measurements were made of intracellular calcium S. salar spermatozoa that were
exposed to Powermilt and to zero calcium Powermilt. Subsequentstily, a representative
trace was used to examine how samples loaded with the probe, present an increase
in the uorescence signal when exposed to normal Powermilt, reecting an increase
in intracellular calcium [Fig. 3(a)]. A second application of Powermilt resulted in a
(b)
5
4
2
Calcium RFU normalized (%)
0
Control
Activated
Activated II
1
3
(d)
1·5
1·0
Calcium RFU normalized (%)
0·0
Control
Post activation
Aging
0·5
(c)
5
4
2
Calcium RFU normalized (%)
0
Control
Zero calcium
Calcium add
1
3
(a) Activated
II Activated
Activated
100 s
1000 RFU
Zero calcium
Without calcium
With calcium
*
*
*
**
F. 3. Effects of activation on intracellular calcium levels in Salmo salar spermatozoa. (a) Traces of calcium
in spermatozoa loaded with a Fluo-4calcium probe under different conditions, upper panel with calcium;
lower without calcium. (b) Mean ±.. (n=3) intracellular calcium content, expressed as relative reference
units (RFU), under different conditions. *, P<0·05, ANOVA comparison with control. RFU, relative uo-
rescence units. (c) Mean ±.. (n=3) intracellular calcium content, expressed as RFU, upon spermatozoa
activation, in the presence and absence of extracellular calcium. *, P<0·05, ANOVA comparison with con-
trol. (d) Mean ±.. (n=3) calcium, expressed as RFU, in control aged spermatozoa; the insets are images
of cells loaded with the probe. *, P<0·05, ANOVA comparison with control.
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
8J. PARODI ET AL.
smaller increase in intracellular calcium concentration. The second application was
applied directly to the sample, without washing or making any changes. In order to
describe a synergistic effect on the activation, more Powermilt was added in a second
addition and calcium levels were observed [Fig. 3(a)].
An increase in calcium was expected, but no change was observed in intracellular
calcium as a second stimulus was used [Fig. 3(b)]; therefore, a synergistic effect in these
samples cannot be recognized. Interestingly, the lower trace recorded, shows the effects
of activation of sperm when a solution of zero calcium Powermilt is used [Fig. 3(a)].
Where an increase in the uorescence signal was still produced, however it occured
at a level lower than the initial increase observed in the normal Powermilt, suggesting
a minor effect when there was no calcium extracellular solution. This is similar to
the second activation, suggesting that activation effects are partially independent of
extracellular calcium [Fig. 3(b)]. When the spermatozoa were activated using normal
Powermilt, a rise in signalling was observed after the rst stimulus, showing a complete
response that is similar to the values observed in the upper panel [Fig. 3(c)]. If the
decrease in calcium levels may reect a loss of sperm function, then it is important
to preserve calcium levels in stored aged milt. The results, obtained by measuring the
average calcium content of isolated cells (10 cells by eld, select 10 different elds) that
were activated in solution with calcium for 5 min after 20 days of storage. The complex
results suggest nely tuned regulation of the calcium ion in S. salar spermatozoa. This
method maintains intracellular calcium storage and functionality of these cells and is
required for calcium entry regulation [Fig. 3(d)].
DISCUSSION
The S. salar spermatozoa sample exhibited low rates of mortality when maintained
in Stopmilt solution at 4∘C, shaken and kept under well oxygenated conditions. More-
over, 50% of motility was preserved for the rst 5 days. This study considered whether
these results were related to the differences in the calcium levels by preserving S. salar
spermatozoa and measuring intracellular calcium levels at different points in time.
Calcium movement correlates with physiological changes in different cell models.
In S. salar spermatozoa, extracellular signalling induces changes to increase ion
permeability, thereby increasing motility (Cosson et al., 1989). The observations here
indicate that the level of intracellular calcium decreases during storage and that after
5 days, the reduction in basal calcium content correlates with a decline in spermatozoa
motility. The patterns shown can be explained by the fact that these spermatozoa are
physiologically suppressed when kept under controlled temperatures and constant oxy-
genation. Such conditions could account for the ability of the milt dilution to suffer low
mortality and maintain intracellular calcium levels for longer periods of time (Ubilla &
Valdebenito, 2011). In mammalian spermatozoa, activated cells produce a large num-
ber of metabolites that can alter their properties (Kumaresan et al., 2012). Salmo salar
spermatozoa seem to maintain their function over time. When activated, they exhibit a
short duration of activity during which they increase their intracellular calcium levels.
A short secondary stimulus, however, does not generate the same type of change. It
is lower but still non-signicant and no synergism on calcium entry were observed.
These observations support the possibility that S. salar spermatozoa, upon nishing
the motility state, when the spermatozoa stop moving, do not regulate calcium, leading
© 2016 The Fisheries Society of the British Isles, Journal of Fish Biology 2016, doi:10.1111/jfb.13245
ROLE OF CALCIUM ON S. SALAR SPERMATOZOA 9
to the possibility of spermatozoa sustaining a second motility period (Christen et al.,
1987). This idea is demonstrated by the small addition of calcium described in the sec-
ond application. No signicant change was observed. When given the same stimulus in
a calcium-free extracellular solution, the spermatozoa showed a small but signicant
increase in intracellular calcium, which suggests that the extracellular stimulus changes
the intracellular calcium content through a mechanism that does not completely depend
on extracellular calcium. In a previous report on spermatozoa from marine teleosts,
Oda & Morisawa (1993) suggested that intercellular calcium are important in initiating
spermatozoa motility, but not did not participate in maintaining spermatozoa motility.
Recent reports indicate that when intracellular calcium is depleted, S. salar spermato-
zoa are unable to activate, remaining immobile (Takei et al., 2012).
In conclusion, this study indicates the existence of a calcium regulation mechanism in
spermatozoa that is preserved over time in a quiescent physiological time (not motile)
in a low metabolic condition. Although it is not yet know what this mechanism is, it
causes the loss of both intracellular calcium and the spermatozoa’s ability to respond
to an activating stimulus. Supporting this idea, when spermatozoa are incubated in
Powermilt, the cells are emptied of intracellular calcium storage. Similarly, non-motile
aged spermatozoa (ageing spermatozoa) lack intracellular calcium and do not respond
to activation stimuli. Thus, S. salar spermatozoa preserved under controlled temper-
atures and oxygenated conditions maintain their physiological properties for several
days, although the capacity to measure fertility is currently not available. It can also
be noted that S. salar spermatozoa possess intracellular calcium stores that decrease
after long-term storage and that this has a negative effect on spermatozoa performance.
These ndings provide insight into how spermatozoa should be handled in vitro, sug-
gesting new avenues of research and improving the understanding of how calcium
affects the management of sh spermatozoa.
This study was funded by a grant (MECESUP-UCT 0804) awarded to J.P. and partial support
from grant 412-455 ‘Fondo de investigación Interna’. We would like to thank R. Saez for his
assistance in the translation and corrections and http://www.journalrevisions.com/ for the nal
edition. The milt samples, Stopmilt and Powermilt solution were provided by I. Valdebenito,
from BIOACUI-UCT.
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