Reprod Dom Anim 41, 241–246 (2006); doi: 10.1111/j.1439-0531.2006.00676.x
ISSN 0936-6768
The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red
Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads
MC Esteso1,2, MR Fernández-Santos1,2, AJ Soler1, V Montoro1, A Quintero-Moreno3 and JJ Garde1,2
1
Grupo de Biologı´a de la Reproducción, Instituto de Investigación en Recursos Cinegéticos, IREC, (CSIC, UCLM, JCCM), Campus Universitario,
Albacete, Spain; 2Sección de Recursos Cinegéticos y Ganaderos, IDR, (UCLM), Albacete, Spain; 3Unidad de Investigación en Producción Animal,
Facultad de Medicina Veterinaria, Universidad de Zulia, Maracaibo, Venezuela
Contents
Computer-automated sperm-head morphometry was used in
this study to determine the effects of cryopreservation on red
deer sperm-head morphometry. Epididymal sperm samples
were collected from 40 mature stags and were divided. One
portion was diluted at room temperature in a Tris-citrate egg
yolk medium, containing 6% glycerol. A microscope slide
was prepared from single extended sperm samples prior to
freezing. The remainder of each sample was frozen in
nitrogen vapours. After thawing, sperm smears were prepared
as described above. All slides were air dried and stained with
Hemacolor. The sperm-head dimensions for length, width,
area, perimeter and shape factor (length/width), for a
minimum of 135 spermatozoa were determined for each slide
by means of the Sperm-Class Analyser (SCA). Firstly, our
results show that cryopreservation substantially reduced
(p < 0.001) sperm motility and plasma membrane and
acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the
companion extended samples for area (32.05 lm2 vs
32.56 lm2; p < 0.05), length (8.46 lm vs 8.53 lm;
p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001)
for all stags. These differences were found within 29 of 40
stags (75%) for at least three of the morphometric
parameters. The individual variability (CV) of sperm head
measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head
measurements after cryopreservation for area (r ¼ )0.465),
width (r ¼ )0.483) and perimeter (r ¼ )0.375). Thus, the
lower the sperm head variability in the extended samples, the
greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be
a good indicator of sperm freezability.
Introduction
The routine evaluation of semen, including normal
sperm morphology assessment, has long been employed
to evaluate the effects of freezing-thawing procedures on
sperm cryosurvival. Poor semen morphology is an
important indicator of decreased fertility in men (Kruger et al. 1993); stallions (Jasko et al. 1990) and bulls
(Sekoni and Gustafsson 1987). Sperm head abnormalities have been associated with early embryonic loss,
lowered fertility and embryo quality (DeJarnette et al.
1992) and reduced capacity to bind to the ovum (Kolt
and Handel 1987). Although normal sperm morphology
may be an indicator of the fertility potential of a given
male, correlations have been based on subjectively
performed analyses. However, large variations between
technicians and laboratories in the subjective evaluation
of semen characteristics are known to exist (Saacke
1982), making accurate interpretation of the resulting
data difficult.
The need for accurate objective assessment of sperm
morphology has led to the development of computerassisted sperm morphometry analysis (ASMA) (Katz
et al. 1986; Pérez-Sánchez et al. 1994). The precision of
the ASMA system has been utilized to detect morphometric differences in sperm head dimensions of fertile
and subfertile males (Casey et al. 1997) as well as
subtle changes in human sperm head morphometry
because of toxicant exposure when no morphological
differences were detected by manual assessment (Davis
et al. 1993). Previous studies utilizing ASMA have also
demonstrated that cryopreservation affects head
morphometry of bull (Gravance et al. 1998), human
(Thompson et al. 1994), stallion (Arruda et al. 2002)
and dog (Rijsselaere et al. 2004) cryopreserved spermatozoa. In these studies, sperm heads were significantly smaller in cryopreserved spermatozoa than in
fresh-extended sperm. The differences in morphometric
dimensions between fresh and cryopreserved spermatozoa have been explained by several possible mechanisms including osmotic changes, acrosome damage
and alterations in chromatin condensation (Gravance
et al. 1998). These observations, suggest that sperm
head dimensions of individual sperm samples, may be
an indicator of sperm cryosurvival.
Thus, adopting methods previously utilized in other
species (Sancho et al. 1998) and in red deer by us
(Esteso et al. 2003), our objective was to determine the
effects of cryopreservation on red deer sperm-head
morphometry. To achieve this goal, we firstly evaluated
the effects of cryopreservation on sperm motility and
on acrosome and membrane integrities. Secondly, we
evaluated the effects of cryopreservation on epididymal
red deer sperm head size. In relation with this second
general aim, we also carried out this study: (i) to
determine whether the effects vary between individual
stags and (ii) to determine which sperm head morphometric measurements, if any, are associated with
changes that occur to the sperm during freezing and
thawing.
Material and Methods
Materials
With the exception of DPX (Fluka, Madrid, Spain), all
other chemicals were of reagent grade and were
purchased from Sigma or Merck (both of Madrid,
Spain).
2006 The Authors. Journal compilation 2006 Blackwell Verlag
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MC Esteso, MR Fernández-Santos, AJ Soler, V Montoro, A Quintero-Moreno and JJ Garde
Preparation of testes and collection of epididymal
spermatozoa
For this study, we used spermatozoa recovered from the
epididymides of 40 mature stags (age >4 years, weight
>140 kg) that were legally culled and hunted in their
natural habitat during the rutting season (September to
November). The hunting of stags was performed in
accordance with the harvest plan of each game reserve.
The harvest plans were made following Spanish Harvest
Regulation, Law 2/93 of Castilla-La Mancha, which
conforms to European Union Regulation. Immediately
upon removal, the testes with attached epididymides
were placed into plastic bags and transported to the
laboratory at room temperature (approximately 20C)
within 2 h after being removed. Samples were processed
as soon as they arrived at the laboratory. Epididymal
spermatozoa were collected as described by Soler et al.
(2005). Epididymal contents from both testicles of an
individual stag were pooled for processing, because an
ejaculate contains a mixture of spermatozoa from both
testicles.
Semen processing and cryopreservation
The sperm mass was diluted at room temperature to
a final sperm concentration approximately 400 ·
106 sperm/ml with a Tris-citrate-20% egg yolk medium,
containing 6% glycerol (Fernández-Santos et al. 2005).
At this point, sub-samples were taken for sperm head
morphometric dimensions evaluation. Then, the sperm
diluted was placed in a 15-ml centrifuge tube (Iwaki,
Tokyo, Japan) and was slowly cooled to 5C. For it, the
tubes were placed in a beaker with water (75 ml at room
temperature) and transferred to a refrigerator at 5C.
Cooling down to 5C lasted for approximately 1 h and
then extended samples were held for equilibration at
that temperature for 2 h more. After the equilibration of
the diluted sperm samples, the extended sperm was
loaded into 0.25 ml plastic straws. Immediately, the
straws were frozen in nitrogen vapours, 4 cm above the
surface of the liquid nitrogen, for 10 min and then
plunged into liquid nitrogen. The straws remained for a
minimum period of 1 month in liquid nitrogen before
thawing was carried out.
Frozen semen was thawed in a water bath (37C) for
20 s and the content of the straws poured into a glass
tube. Samples were evaluated for motility, viability, and
acrosome and membrane integrities after 5 min of
incubation at 37C, using the methods described below.
At thawing, subsamples were also taken for sperm head
morphometric evaluation.
Semen evaluation
Sperm concentration and subjective scores of motility
were assessed shortly after collection. Sperm concentrations of the original suspensions were determined using
a haematocytometer. Percentage of individual motile
sperm (motility) was noted and quality of motility was
assessed using a scale of 0, lowest, to 5, highest. A Sperm
Motility Index (SMI) was calculated ¼ [% individual
motility + (quality of motility · 20)] · 0.5. The sperm
suspension was also used to assess acrosome integrity
and viability. Acrosomal integrity was evaluated after a
1 : 1 dilution in 2% glutaraldehyde in 0.165 M cacodylate/HCl buffer (pH 7.3). The percentage of spermatozoa with intact acrosomes [% normal apical ridge
(NAR)] was assessed by phase-contrast microscopy.
Only samples with an initial sperm motility and NAR
>65–70% were used for freezing.
In addition, samples were taken to assess the membrane integrity by means of the hypo-osmotic swelling
(HOS) test. Plasma membrane functionality was
assessed using a HOS test as described by Garde et al.
(1998). The sperm membrane was considered functional
in cases where the sperm tail was coiled and the result
was expressed as HOS (%).
Membrane integrity (viability) was also evaluated by
using a nigrosin-eosin (NE) stain. The NE stain was
prepared according to the method by Soler et al. (2005).
Live spermatozoa remained unstained, while dead cells
were dull pink. The percentage of live spermatozoa was
expressed as viability (%).
Two hundred sperm cells were assessed in each sample
and for each sperm evaluation technique. Additionally,
slides of extended and thawed semen were prepared
from each sample for sperm head morphometric characterization.
Morphometric analysis of sperm heads
Microscopic slides were prepared from each extended
(upon dilution) and cryopreserved sample by placing
5 ll of the sperm samples on the clear end of a frosted
slide and dragging the drop across the slide. Semen
smears were air dried and stained using a Hemacolor
(Merck) procedure, originally described for staining of
ram (Sancho et al. 1998) sperm heads; and recently
adapted by our group to red deer spermatozoa (Esteso
et al. 2003). Stained sperm samples were permanently
mounted to the slide with a coverslip and dibutyl
phthalate xylene (DPX).
Stained slides were used to perform ASMA using the
morphometry module of a commercially available
system (Sperm-Class Analyzer; Microptic, Barcelona,
Spain). The machine was equipped with a Nykon
(Labophot-2, Tokyo, Japan) microscope with a ·60
bright-field objective and a Sony video camera (CCD
AVC-D7CE; Sony Corporation, Tokyo, Japan) connected to a Pentium 950 MHz processor. The illumination
source was centred and the intensity of the bulb and the
gain and offset of the camera were standardized for all
samples. The configuration of the computer system
included a PIP-1024 B video digitizer board (Matrox
Electronic Systems Ltd., Quebec, Canada), the sperm
image analysis software and a high-resolution assistant
monitor Sony Triniton PVM-1443MD (Sony Corporation). The array size of the video frame recorder was
512 · 512 · 8 bits, digitized images were made up of
262 144 pixels (picture elements) and 256 grey levels.
Resolution of images was 0.15 and 0.11 lm per pixel in
the horizontal and vertical axes, respectively.
The morphometric dimensions for area (A), perimeter
(P), length (L), width (W), and shape factor (L/W) were
2006 The Authors. Journal compilation 2006 Blackwell Verlag
Sperm Head Morphometry and Cryopreservation
acquired for 140–150 images. Acquiring 140–150 images
assures that a minimum of 135 properly measured sperm
heads are analysed after improperly measured sperm
heads are deleted from the analysis. The sperm cells were
randomly selected for the morphometric analysis. The
measurements of each individual sperm head from each
stag and sperm treatment were saved in an Excel
(Microsoft Corporation, Redmond, Washington,
USA) – compatible database by the software for further
analysis.
Statistical analysis
Statistical analyses were performed using SPSS for
Windows, version 11.0 (SPSS Inc, Chicago, IL, USA).
The effects of cryopreservation on sperm motility,
viability, acrosome and membrane integrities and sperm
head morphometric dimensions were compared within
and across stags by General Linear Model analysis of
variance (GLM-ANOVA) using a split plot design. Stags
served as the main plot and sperm cryopreservation step
(pre-freezing or post-thawing) served as the subplot.
Group differences were compared by Fisher’s Least
Significant Differences test. Effects were considered
significant at p < 0.05. The effects of sperm treatment
on sperm-head morphometry within stags, where effects
were observed, were compared by Student’s t-test. The
within analysis variation (CV) and between stags variation within morphometric measures were compared
between sperm treatments by the Mann–Whitney twosample test. Correlations between sperm head measurements (values and CVs) and the standard semen
parameters (motility, viability, HOS and NAR) after
cryopreservation were performed by Spearman’s rank
correlation coefficients. Data that did not follow a
normal distribution were transformed.
Results
In this work, epididymal spermatozoa from 40 stags
were frozen and thawed. Because we used epididymal
spermatozoa for this experiment, it was only possible to
make single observations for each stag. After freezing
and thawing, a decrease (p < 0.001) in all routine sperm
parameters was observed. Thus, SMI decreased from
74.51 ± 2.2% in extended fresh samples to
63.88 ± 1.7% in thawed samples. Spermatozoa with
normal acrosomes decreased (p < 0.001) from
82.15 ± 2.3% in extended samples to 62.79 ± 2.1%
in thawed samples. The results found for the other
parameters (HOS test and viability) were similar if not
identical (data not shown).
The results of the comparison of sperm heads
dimensions between extended and cryopreserved samples are summarized in Table 1. This table shows the
mean sperm head measurements and the CVs between
stags for extended and cryopreserved samples. A total of
5,451 property digitized spermatozoa for extended
samples, and 5,416 for cryopreserved samples were
analysed. There were no differences in the number of
properly analysed sperm heads between extended and
cryopreserved samples (data not shown). The values for
all measures of sperm heads dimensions were deter-
243
Table 1. Mean sperm head measurements of area (A), perimeter (P),
length (L), width (W) and shape factor (L/W) for extended and
cryopreserved sperm samples from 40 stags
Parameters
Sample
2
A (lm )
P (lm)
L (lm)
W (lm)
EXT
32.56 (9.6)a 30.05 (7.9)a 8.53 (6.6)c 4.63 (7.2)a
CRYO 32.05 (9.6)b 30.00 (7.7)a 8.46 (6.8)d 4.63 (7.2)a
Shape factor
N
1.849 (8.4)c
1.833 (8.8)d
5,451
5,416
EXT, extended sperm sample; CRYO, cryopreserved sperm sample; N, total
number of sperm counted per treatment.
Coefficients of variation (% CV) between stags are shown in parentheses.
Values with different superscript letters in the same column were significantly
different. a, bp < 0.05, c, dp < 0.0001.
mined to be normally distributed by Kolmogorov–
Smirnov normality test. Analysis of variance showed a
significant (p < 0.001) within-stag effect on sperm head
morphometric dimensions in the extended and cryopreserved samples. Besides, significant effects (p < 0.05) of
cryopreservation were found among stags on sperm
head dimensions. In this sense, sperm heads were
smaller in cryopreserved samples than in the companion
extended samples for area (32.05 lm2 vs 32.56 lm2,
p < 0.05), length (8.46 lm vs 8.53 lm, p < 0.0001)
and shape factor (1.833 vs 1.849, p < 0.0001) between
all males. The variation (per cent CV) of the mean
dimensions across all stags ranged from 6.6% (L) to
9.6% (A) for extended samples, and 6.8% (L) to 9.6%
(A) for cryopreserved samples (Table 1).
Our results also revealed that all sperm head measurements were significantly (p < 0.001) affected by the
interaction individual factor-sperm treatment. In this
sense, no significant cryopreservation effect was found
between the extended samples and the thawed ones for
sperm head dimensions within stags for all males. Thus,
in 29 of the 40 stags (75%), differences (p < 0.05) were
observed in at least three of the five morphometric
parameters between extended and cryopreserved samples. The per cent change in sperm head measurements
from extended and cryopreserved samples of the eleven
stags that showed no differences (ND) and the 29 males
where differences (DIF) occurred, were different
(p < 0.0001) for area ()5.6 vs 3.53%), length ()2.1 vs
2.5%) and width ()3.7 vs 1.8%). No significant
differences in any sperm head dimensions were detected
in cryopreserved samples when the ND group of stags
was compared with the DIF group. Contrary, significant
differences were found in sperm head dimensions in
extended samples when the ND group was compared
with the DIF group for area (ND ¼ 31.43 lm2 vs
DIF ¼ 33.13 lm2; p ¼ 0.026) and length (ND ¼
8.41 lm vs DIF ¼ 8.66 lm; p ¼ 0.024). No significant
differences in the variability of any sperm head dimensions were detected in extended or cryopreserved samples when the ND group of stags was compared with the
DIF group. Finally, at thawing SMI (ND:
68.87 ± 3.1% vs DIF: 61.59 ± 2.0%, p ¼ 0.06) and
sperm viability (ND: 70.25 ± 3.1% vs DIF:
63.22 ± 2.1%, p ¼ 0.06) tended to be higher for the
stags that did not incur changes in the morphometric
parameters of the sperm head than for those did it, but
differences were not statistically significant.
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MC Esteso, MR Fernández-Santos, AJ Soler, V Montoro, A Quintero-Moreno and JJ Garde
Table 2. Mean within analysis coefficients of variation (%) for area
(A), perimeter (P), length (L), width (W), and shape factor of sperm
heads of extended and cryopreserved sperm samples from 40 stags
10
Sample
A
P
L
W
Shape factor
N
EXT
CRYO
6.20a
7.02b
6.20a
6.48a
5.12c
5.50d
5.41a
5.50a
6.85a
6.80a
5,451
5,416
EXT, extended sperm sample; CRYO, cryopreserved sperm sample; N, total
number of sperm counted per treatment.
Values with different superscript letters in the same column were significantly
different. a, bp < 0.005, c, dp < 0.05.
CV (%)
Parameters
8
6
Besides, significant differences (p < 0.05) in the
means within analysis CVs were found between the
extended and cryopreserved samples for area and length
among stags (Table 2). In this sense, the variability of
sperm heads measurements was lower in extended semen
samples than in cryopreserved ones for area (6.20% vs
7.02%, p < 0.005) and length (5.12% vs 5.50%,
p < 0.05) when all samples were analysed. Finally, the
results of the ANOVA procedure revealed that within
analysis CVs were significantly (p < 0.001) affected by
the interaction between individual factor and sperm
treatment. Overall, the results from the 40 stags showed
a similar pattern of response, with higher (p < 0.05)
variability (per cent CV) in cryopreserved samples than
in the extended ones. However, when we analyses only
the 11 stags of the ND group, no significant differences
(p > 0.05) in the means within analysis CVs were found
between the extended and cryopreserved samples for any
sperm head measurements.
The per cent difference in individual parameters of
head measurements of spermatozoa from extended and
cryopreserved samples for all stags was negatively
correlated (p < 0.005) with the CVs of the corresponding measurements for area (r ¼ )0.465), width (r ¼
)0.483) and perimeter (r ¼ )0.375) of the initial extended sample. These results are showed in Fig. 1 for sperm
head area. These relations were not found with the CVs
of the sperm heads dimensions for the cryopreserved
samples.
Finally, the per cent difference in individual parameters of head measurements of spermatozoa from
extended and cryopreserved sperm samples for all stags
was not correlated (p > 0.05) with any sperm routine
parameters determined in extended or cryopreserved
samples. Besides, the variations (CV) of sperm head
dimensions of spermatozoa from extended samples were
not related to standard sperm parameters determined in
cryopreserved samples when all stags were analysed.
Discussion
Firstly, we found a significant effect of cryopreservation
on the head morphometry of red deer epididymal sperm,
across a population of 40 stags. Sperm head measurements of cryopreserved samples were significantly lower
than those of the extended samples for area, length and
shape factor across all stags. Our results, thus, indicate
that the extended spermatozoa, in addition to being the
ones having higher sperm quality (determined by SMI,
–10
0
10
Change (%)
Fig. 1. The variability (% CV) of sperm head area in extended samples
(unfrozen) is plotted as a function of the per cent difference in the
corresponding measurement of head spermatozoa from extended and
cryopreserved samples (solid points). The correlation coefficient is r ¼
)0.465 (n ¼ 40; p < 0.005)
NAR, viability and HOS test), are also those with larger
sperm heads, comparing with cryopreserved spermatozoa. Secondly, our results show too that, whereas
significant freeze/thaw effects were found in the morphometric measurements across the population of 40 stags,
significant differences were found only within some
stags. Indeed, a low number of the stags (25%) in this
study did not incur changes in the morphometric
parameters of the sperm head. The detection of freeze/
thaw effects within individual stags indicates that some
individuals may be more sensitive to cryopreservation.
Besides, sperm head dimensions from extended samples
were significantly lower in those samples that showed no
changes in measurements. Finally, at thawing, SMI and
sperm viability tended to be higher for the stags that did
not incur changes in the morphometric parameters of
the sperm head than for those that did it, although the
differences were not statistically significant. Therefore,
our results indicate that extended sperm samples with
lower initial dimensions of sperm heads, in addition to
being the ones where morphometric changes in sperm
heads did not occur after cryopreservation, are also
those with a tendency to higher sperm quality at
thawing, with the opposite being true for spermatozoa
with larger initial sperm head dimensions.
In relation with the effects of cryopreservation on
sperm head morphometric measurements, our results
agree with earlier findings reported for bull (Gravance
et al. 1998), human (Thompson et al. 1994), stallion
(Arruda et al. 2002) and dog spermatozoa (Rijsselaere
et al. 2004), in which sperm head dimensions (length,
width, area and perimeter) were smaller in cryopreserved samples than in fresh extended samples. These
differences in morphometric dimensions between fresh
2006 The Authors. Journal compilation 2006 Blackwell Verlag
Sperm Head Morphometry and Cryopreservation
and cryopreserved spermatozoa have been explained
proposing several possible mechanisms, including osmotic changes, acrosome damage and alterations in chromatin condensation (Royere et al. 1988; Gravance et al.
1998; Love and Kenney 1998; Blottner et al. 2001).
However, our results only partially agree with those
earlier reported for the effects of cryopreservation on
head morphometry of goat spermatozoa (Gravance
et al. 1997). These authors found no overall effect of
cryopreservation on goat sperm head morphometry, but
they observed an effect within a limited number of
bucks. Contrasting results may be because of a certain
species specific sensitivity for the freezing process or to
different cryopreservation protocols (i.e. glycerol levels,
freezing and thawing rates), resulting in a different effect
on the post-thaw sperm head characteristics (Thompson
et al. 1994; Gravance et al. 1998).
Our results also show that the effect of cryopreservation on red deer sperm heads varies among individuals.
However, in contrast to the previous findings of
Gravance et al. (1997), the majority (75%) of the stags
in this study incurred changes in the sperm head
dimensions during the cryopreservation process. The
average per cent change across all stags ranged from
0.5% for width to 12% for area. Although the average
changes in dimensions were <12% for all measurements, they were still found to be significantly different,
because of the precision of the SCA. This method of
analysis was able to detect the very small changes in
morphometric dimensions between extended and cryopreserved sperm heads.
The CVs (variation) of sperm head measurements
within and across stags (Tables 1 and 2), indicate that
sperm head measurements from different stags are
extremely heterogeneous. In fact, at least as heterogeneous as the sperm population within a sample of a
given stag. Besides, our results show that the variability
of sperm heads measurements was lower in the extended
semen samples than in the cryopreserved ones for area
and length when all samples were analysed. However,
when this effect was independently studied in the two
groups of stags (ND and DIF), these differences were
only significant for the DIF group. These results suggest
that sperm head area and length from different stags are
more heterogeneous in the cryopreserved samples than
in the extended ones, especially in the samples from DIF
stags. Thus, our results show that the variability of
sperm head dimensions between cryopreserved and
extended samples was only significantly different in the
samples that showed changes in measurements during
the cryopreservation.
Whereas cryopreserved sperm samples showed no
significant differences in the sperm head morphometric
dimensions of the ND and DIF populations, sperm head
dimensions from extended samples were significantly
lower in the samples that showed no changes in
measurements. Taken together, our results indicate that
extended sperm samples with lower initial dimensions of
sperm heads, in addition to being the ones which less
change their sperm head measurements between dilution
and cryopreservation, are also those with lower variability of sperm head dimensions at thawing, with the
opposite being true for spermatozoa with higher initial
245
dimensions of sperm head. Thus, these two groups of
males differ in the dimensions of their sperm heads, and
it is possible that these differences have an impact on
sperm water volume and membrane permeability to
water and cryoprotectants and, in turn, on sperm
freezability (Curry 2000). Therefore, the lower the
sperm head dimensions in the extended samples, the
greater the sperm cryoresistance. Our results also show
that the change (%) in sperm head measurements from
extended and cryopreserved samples for all stags was
negatively correlated with the CVs of the corresponding
measurements for area, width and perimeter of the
extended sperm heads. These negative correlations
indicate that, regardless of the male group (ND or
DIF), as the variability of the extended samples
decreased, the per cent difference in individual parameters of sperm head measurements from extended to
cryopreserved samples increased in a correlation fashion. Indeed, our results show that the greater the sperm
head area variability (CVs) in extended samples (Fig. 1),
the lower the sperm cryodamage (as revealed by the per
cent difference from extended to cryopreserved samples). Therefore, the sperm head morphometric variability of an extended sample can be a good indicator of
the sperm cryosurvival of the same sample. To the best
of our knowledge, this is the first report demonstrating
that sperm head morphometric dimensions and heterogeneity of individual extended samples may be used as
good indicators of sperm resistance to freezing-thawing
process.
The examination of post-thaw spermatozoa with
techniques such as partitioning in aqueous two phases
systems to detect subtle differences in surface properties
has demonstrated that heterogeneity is severely diminished after the freeze-thaw process (Ollero et al. 1998).
Contrary, our results show that significant differences in
the means within analysis CVs were found between the
extended and cryopreserved samples for area and length
among stags, being the variability (heterogeneity) of
sperm head measurements lower in the extended samples than in the cryopreserved ones when all samples
were analysed. These apparently conflicting findings
could be accounted for in one of two ways. One of the
explanations could be that the use of two different
techniques to evaluate sperm heterogeneity might differentially affect results. Alternatively, when we analysed the effect of cryopreservation in the CVs separately
for the two groups of males (ND and DIF), we only
found significant differences (p < 0.05) in the means
within analysis CVs between the extended and cryopreserved samples for DIF group. These results indicate
that the variability of sperm head dimensions from
cryopreserved samples was significantly higher than that
from extended samples only in the samples that showed
changes in measurements (i.e. samples that were more
affected by cryopreservation). Therefore, in these stags
the freeze-thaw process might affect an higher population of cells than in ND stags, originating a higher rate
and extent of cellular damage. This higher degree of
sperm lesion might be the responsible of the higher
variability of sperm head dimensions from cryopreserved samples in this group of stags (DIF), giving the
appearance of a more heterogeneous population.
2006 The Authors. Journal compilation 2006 Blackwell Verlag
246
MC Esteso, MR Fernández-Santos, AJ Soler, V Montoro, A Quintero-Moreno and JJ Garde
In conclusion, sperm head morphometric dimensions
were significantly smaller in the cryopreserved samples
than in the extended ones, across a population of 40
males. The impact of the effects was variable across
stags, with only 25% of the stags showing no significant
change in morphometric dimensions. In addition, sperm
freezability tended to be higher for the stags that did not
incur changes in the morphometric parameters of the
sperm head than for those did it, but the differences were
not statistically significant. The variability (CV) of
sperm head measurements from extended samples was
negatively correlated with the per cent of change in
sperm head measurements after cryopreservation. It
appears that the variability of the sample prior to
cryopreservation may be predictive of subsequent changes. Future work will utilize ASMA to identify possible
relationships between sperm head dimensions and the
fertility.
Acknowledgements
The authors thank Ibercaza and Cinegética Los Azores for their
collaboration in the collection of the samples used in this work. This
study was sponsored by Grant PBC-02–011 from the Consejerı́a de
Ciencia y Tecnologı́a de la Junta de Comunidades de Castilla-La
Mancha (JCCM), Spain. A. Quintero-Moreno was sponsored by
research funds of the International Cooperation Office from the
UCLM (Spain). We thank Dr F. Martinez-Pastor for his suggestions
and review of the manuscript.
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Submitted: 24.10.2005
Author’s address (for correspondence): Dr José Julian Garde, Grupo
de Biologı́a de la Reproducción, IREC, Campus Universitario, sn,
02071, Albacete, Spain. E-mail: julian.garde@uclm.es
Present address: AJ Soler, CERSYRA de Valdepeñas, Consejerı́a de
Agricultura JCCM, Valdepeñas, Ciudad Real, Spain.
2006 The Authors. Journal compilation 2006 Blackwell Verlag