Post on 27-Jan-2023
Seasonal functional relevance of sperm characteristics
in equine spermatozoa
S. Gamboa a,b,*, A.S. Rodrigues b, L. Henriques a, C. Batista a, J. Ramalho-Santos b
a Animal Reproduction Laboratory, Department of Zootechnic Sciences, Agricultural School,
Polytechnic Institute of Coimbra, Bencanta, Coimbra, Portugalb Center for Neuroscience and Cell Biology, Department of Life Sciences, Faculty of Science and Technology,
University of Coimbra, Coimbra, Portugal
Received 1 August 2009; received in revised form 3 November 2009; accepted 22 November 2009
Abstract
A group of stallions with different reproductive indexes were used to study seasonal variations in sperm quality (Equus caballus).
Semen samples were collected from late September to July and analyzed according to four seasonal periods: late September–
December, January–March, late March–May, and June–July. Parameters monitored included sperm concentration, sperm motility,
sperm morphology, sperm viability, acrosomal status, plasma membrane stability, and sperm mitochondrial membrane potential.
Overall, seminal parameters monitored are affected mostly by time period, followed by animal and lastly by fertility, stressing the
importance of individual variations in out-bred animal models. The analysis of multiple ejaculates from the same animals showed
clear seasonal-based differences (P < 0.05) with poor semen quality in winter and a noticeable improvement in sperm quality with
increasing photoperiod. Better semen quality was observed between late March and May. Interactions between month period,
animal, and fertility were evident (P < 0.05) for sperm concentration, head and tail sperm anomalies, and acrosomal integrity. Thus,
it may be advisable to adjust the use of stallion semen according to seasonal variations.
# 2010 Elsevier Inc. All rights reserved.
Keywords: Equine; Mitochondrial function; Motility; Semen; Spermatozoa
www.theriojournal.com
Available online at www.sciencedirect.com
Theriogenology 73 (2010) 950–958
1. Introduction
Similarly to other mammals, equines restrict their
reproductive efforts to the time of the year when
conditions are most favorable for the successful
weaning of offspring [1]. The seasonal change in day
length is the main, though not the only, cue responsible
for the circannual rhythm of reproductive activity [2].
On the whole, the fertile period takes place from April
to September in the Northern Hemisphere [3]. However,
* Corresponding author. Tel.: +351 239 802940;
fax: +351 239 802979.
E-mail address: scgamboa@esac.pt (S. Gamboa).
0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2009.11.023
in the majority of equine breeding associations, the
breeding season is officially established from February
to June, forcing breeders to breed mares as early as
possible in the year. To what extent the quality of semen
collected early in the breeding season can affect
reproductive indexes is not known.
In the horse, male gametogenesis, reproductive tract
function, and resulting sperm functionality are likely to
be optimal during the breeding season. Throughout the
winter there seems to be a decrease in sperm production
[4], and seasonal fluctuations have also been observed
for sperm viability and motility [5,6]. According to
Guillaume [7], fertility in stallions may be maintained at
a reduced level outside the breeding season. Moreover,
seasonal breeders from latitudes between 308N and
S. Gamboa et al. / Theriogenology 73 (2010) 950–958 951
408N do not exhibit the same dramatic seasonal changes
in reproductive activity as animals from higher latitudes
[8]. Nevertheless, no data exist on seasonal variation in
semen quality in horses from those latitudes.
The in vivo fertilizing ability of a given sperm
sample is dependent on the interplay of a variety of
attributes representing different features required for
fertility. Sperm motility, in particular, is considered to
be a fundamental laboratory test for assessing an
ejaculate [9]. Further aspects of sperm functionality that
can be probed include sperm nuclear DNA integrity,
acrosomal contents, plasma membrane stability related
to capacitation, and mitochondrial activity in the sperm
midpiece, among others (for a review, see [10]).
In this work, we have attempted to characterize
seasonal variations in equine semen quality, namely
monitoring sperm concentration, motility, morphology
and viability, as well as acrosomal status, plasma
membrane stability, and mitochondrial membrane
potential. Furthermore, and unlike the majority of
previous studies, we have attempted to average out what
sperm characteristics are functionally important by
obtaining a series of samples from the same males
during different seasonal periods.
2. Materials and methods
2.1. Stallions and collections
This study was undertaken using healthy adult male
horses (Equus caballus) from different breeds as follows:
three Puro Sangue Lusitano (PSL), three Sorraia, one
Selle Francais, one Garrano, and one Anglo-Arab.
Animals were housed at the Agricultural School of
Coimbra (408200N, 88410W). The horses were kept
indoors in boxes with straw, and water was freely
available. They were fed three times daily with hay and a
complementary composed food formulated for stallions
and manufactured locally. Semen was routinely collected
(at least two collections/stallion per week) using a
phantom (Hannover model) and an artificial vagina
(INRA model) from late September to July. As stallions
were sexually rested throughout August, before starting
the study one ejaculate was collected daily from each
stallion for 5 d to start frequent semen collections at a
level of daily sperm output (DSO).
2.2. Experimental design
From September to December, we analyzed a mean
of three semen samples/stallion per month (N = 29)
obtained from three stallions to characterize semen
quality during the winter. Of these three stallions, one of
them (a Sorraia horse) was considered to be repre-
sentative (considering seminal characteristics and
reproductive performance levels) for the other two
Sorraia horses used to study semen quality during the
breeding season (late January to early May). The other
two stallions (Garrano and Anglo-Arab) were also part
of the study during the breeding season.
From January to July, we analyzed 139 semen
samples obtained from 7 stallions (2 PSL, 2 Sorraia, 1
Garrano, 1 Selle Francais, and 1 Anglo-Arab). The data
were distributed according to three periods in the
reproductive season as follows: from January to March
20, 41 semen samples were analyzed (2 PSL, 1 Sorraia,
1 Garrano, 1 Selle Francais, and 1 Anglo-Arab); from
March 21 to early May, 39 samples were analyzed (2
PSL, 1 Sorraia, 1 Garrano, 1 Selle Francais, and 1
Anglo-Arab); and from May to July, 30 samples were
obtained (2 PSL, 1 Sorraia, and 1 Garrano).
Given that the stallions used were involved in
artificial insemination breeding programs for several
breeding seasons (officially established from February
to June in Portugal), it was possible to retrospectively
divide them in two groups based on fertility trials
carried out during the breeding seasons: Group A (2
PSL, 1 Selle Francais, and 1 Anglo-Arab), represented
by stallions with high fertility at the end of the breeding
season (81.97% to 89.79%), and Group B (3 Sorraia and
1 Garrano), represented by stallions with low fertility at
the end of the breeding season (0 to 66.67%) (Table 1).
Fertility rates were calculated as previously described
[11].
Unless otherwise stated, all reagents were purchased
from Sigma-Aldrich (St. Louis, MO, USA). All sperm
parameters were easily monitored, and results were
reproducible using blinded multiple observers. In all
analysis involving sperm characteristics, at least 200
cells were counted for each sample. After collection,
semen was immediately analyzed (sperm concentration,
motility, morphology, and pH) as previously described
[11]. Morphology was monitored using India ink
(Pelikan, Barcelona, Spain) as a contrasting stain
[12]. In addition, different parameters were evaluated
by fluorescence microscopy with a HUND H 600 AFL
(Helmut Hund GmbH, Wetzlar, Germany) fluorescence
microscope using distinct probes, as described in the
following sections.
2.2.1. Viability
Viability was analyzed with the Live/Dead Sperm
Viability Kit (Molecular Probes Inc., Eugene, OR,
USA). A cell suspension (20 � 106 sperm/mL in Hanks
S. Gamboa et al. / Theriogenology 73 (2010) 950–958952
Table 1
Fertility indexes obtained for eight stallions used in several breeding seasons.
Stallions (breed)/group Number of
mares breed
Number of
estrous cycles
explored
Number of
fertile cycles
Number of
pregnant mares
Per cycle
fertility, %
Fertility at the end of
the breeding season, %
A (Sorraia) 9 14 4 4 28.57 44.44
B (Anglo-Arab)/Group A 83 105 70 68 66.67 81.97
C (Garrano)/Group B 6 9 4 4 44.44 66.67
D (Sorraia)/Group B 7 18 1 1 5.56 16.67
E (Sorraia)/Group B 6 13 0 0 0.00 0.00
F (PSL)/Group A 42 52 37 37 71.15 89.79
G (PSL)/Group A 19 30 16 16 53.33 84.51
H (Selle-Francais)/Group A 7 16 7 6 43.75 85.71
PSL, Puro Sangue Lusitano breed.
Heppes (HH) solution–1% Bovine Serum Albumin
(BSA)) was first incubated for 5 min at 35 8C with the
membrane-permeant SYBR14 nucleic acid stain at a
final concentration of 6 mM (labels all sperm with green
fluorescence, independently of their viability) and then
further incubated (5 min at 35 8C) with the conventional
dead-cell nucleic acid stain, propidium iodide (PI;
0.48 mM). Sperm samples (5.5 mL) were placed on
glass microscope slides with coverslips, and 200 cells/
ejaculate were counted.
2.2.2. Acrosomal status
Acrosomal integrity was estimated with fluorescein
isothiocyanate (FITC)-labeled Pisum sativum aggluti-
nin (PSA-FITC; Sigma) as described elsewhere [11].
Intact acrosomal contents bind the fluorescent lectin,
and the sperm head is thus stained with a uniform bright
green cap-like signal. Sperm samples (5.5 mL) were
placed on glass microscope slides with coverslips, and
200 cells/ejaculate were analyzed.
2.2.3. Sperm plasma membrane stability
Spermatozoa with a high degree of phospholipids
disorder in the plasma membrane were detected using
merocyanine 540 (M540; Molecular Probes). A final
concentration of 25 � 106 sperm/mL was incubated
with M540 in DMSO (2.7 mM, 30 min at 35 8C). M540
stains preferentially membranes with highly disordered
lipids [13]. Sperm samples (5.5 mL) were placed on
glass microscope slides with coverslips, and 200 cells/
ejaculate were counted. Spermatozoa with unstable
membranes (that bind M540 and are red) were
quantified. Additionally, merocyanine labeling was
also assayed simultaneously with motility, in this case
rendering four distinct sperm subpopulations (labeled/
motile, unlabeled/motile, labeled/immotile, and unla-
beled/immotile).
2.2.4. Mitochondrial membrane potential
Inner mitochondrial membrane potential (DCmit)
was monitored with the cationic dye 5,5’,6,6’-tetra-
chloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanine
iodide (JC-1; Molecular Probes). JC-1 shows fluores-
cence changes associated with the inner mitochondrial
transmembrane potential (IMM) [14], depicting orange-
red fluorescence in fully functional midpiece mitochon-
dria and green fluorescence in cells with lower inner
mitochondrial membrane potential, [15,16]. Semen was
diluted to 20 � 106 cells/mL and then incubated for
20 min at 35 8C with JC-1 in DMSO (2 mM). Sperm
samples (5.5 mL) were placed on glass microscope
slides with coverslips, and 200 cells/ejaculate were
analyzed. Staining patterns in both motile and immotile
sperm were quantified.
2.2.5. Statistical analysis
Multiple analysis of variance (MANOVA) was
performed to study interactions between animals,
fertility, and time period for all variables analyzed.
When no interactions were found, isolated effect of
animal, month period, and fertility group were analyzed
by one-way ANOVA and by multiple comparison tests
(least significant differences; LSD). A significant
difference was reported at P < 0.05, otherwise results
are noted as being non-significant (NS). All statistical
analyses were done using SPSS version 15.0 for
Windows (SPSS Inc., Chicago, IL, USA).
3. Results
All fluorescent assays resulted in clearly distinct
sperm populations that could be easily quantified. In
terms of the fluorescence-based PI/SYBR14 assay,
viability was quantified by classifying sperm as live
(intact cell membranes: green fluorescence) or dead
S. Gamboa et al. / Theriogenology 73 (2010) 950–958 953
Fig. 1. Equine sperm labeled with the fluorescent probes (A) PSA-FITC, (B) M540, and (C) JC-1. (A) Sperm with intact acrosomes were
characterized by a clear labeling of the entire acrosomal region of the sperm head. (B) Sperm with loose plasma membrane lipid packing was stained
with merocyanine, mostly in the head and midpiece regions. (C) Using JC-1, labeled sperm showed a clear midpiece-only staining pattern; the
midpiece was either homogeneously green (low DCmit) or with speckles of yellow/orange/red (high DCmit). Scale bar = 5 mm.
(damaged cell membranes: red and/or green to red
fluorescence). Intact acrosomes were visualized thanks
to a uniform PSA-FITC label in the acrosomal region.
When spermatozoa were stained with JC-1, we could
Table 2
Seminal characteristicsa from stallions at winter and at the beginning (Januar
to July) of the breeding season.
Parameter September to December Januar
Sperm concentration (�106) 228.34 � 77.43y 221.45
Sperm progressive motility (%)b 32.16 � 19.32*y 32.41
Sperm vitality, IP/SYBR14 (%)c 47.37 � 17.67* 60.58
Morphologically abnormal spermd
Head defects (%) 17.19 � 10.65*y 19.44
Midpiece defects (%) 9.88 � 4.70y 7.66 �Tail defects (%) 15.60 � 7.91 8.73 �
Semen pH 7.47 � 0.32 7.55 �Intact acrosomes (%)e 67.36 � 10.21*y 72.21
Total sperm "DCmit (%)f 34.21 � 25.60* 65.20
Total sperm #DCmit (%)f 52.94 � 21.00 29.30
Motile sperm "DCmit (%)f 22.17 � 24.11* 41.33
Immotile sperm "DCmit (%)f 12.05 � 11.70 20.81
Motile sperm #DCmit (%)f 21.98 � 16.47y 16.14
Immotile sperm #DCmit (%)f 30.96 � 24.07* 15.03
No DCmit motile sperm (%)f 12.96 � 13.86* 6.72 �Motile sperm M540+ (%)g 6.04 � 6.43 1.04 �Immotile sperm M540+ (%)g 39.88 � 9.81y 38.07
Motile sperm M540– (%)g 24.58 � 22.48 48.50
Immotile sperm M540– (%)g 29.51 � 23.37 12.38
Total sperm M540+ (%)g 45.68 � 11.57y 39.11
For each time period considered, * denotes differences (P < 0.05) between
Between consecutive time periods, identical symbols (y, z, §) denote no di
The semen samples were examined immediately after collection.a n = 168 semen samples.b Progressive motility sperm observed after collection.c Viable (PI/SYBR14 stained) cells; for each ejaculate, counts were perfd Morphologically abnormal sperm; for each ejaculate, counts were perfoe Acrosome intact (PSA-FITC stained) sperm cells; for each ejaculate, cf Sperm cells stained with JC-1; for each ejaculate, counts were performg Sperm cells stained with M540; for each ejaculate, counts were perform
easily observe that some motile and immotile sperm
were unstained in all samples; four sperm populations
exclusively stained in the midpiece (Fig. 1) were
detected: a green background speckled with orange/red
y to mid-March), middle (mid-March to mid-May), and end (mid-May
y to mid-March Mid-March to mid-May Mid-May to July
� 125.99*y 210.44 � 106.32*§ 164.53 � 63.99§
� 15.97*yz 39.24 � 15.55*z 22.75 � 11.49*
� 17.46z 66.30 � 17.17*z 52.00 � 15.04
� 17.98*y 11.11 � 12.00*§ 10.85 � 7.24§
5.50yz 6.13 � 3.23z§ 6.60 � 4.72§
7.31z 6.21 � 4.28z§ 4.73 � 3.86§
0.36 7.47 � 0.39 7.40 � 0.33
� 11.38y 79.86 � 8.50 88.96 � 7.60
� 16.85* 73.09 � 10.57§ 72.90 � 8.58§
� 15.04 21.81 � 11.13§ 27.10 � 8.58*§
� 19.47*z 48.72 � 18.19*z
� 15.28z 21.39 � 15.33z
� 13.31yz 12.36 � 6.92z
� 11.03z 14.85 � 9.92z
7.33* 2.73 � 3.63*
2.10 1.96 � 3.74
� 21.63y 29.88 � 14.79
� 22.54 49.04 � 21.84
� 8.36 17.72 � 13.78
� 21.42y 32.62 � 15.75*
stallions.
fferences (P < 0.05) for sperm parameters.
ormed on 200 cells.
rmed on 200 cells.
ounts were performed on 200 cells.
ed on 200 cells.
ed on 200 cells.
S. Gamboa et al. / Theriogenology 73 (2010) 950–958954
mitochondria (high DCmit) in both motile and immotile
sperm and a homogeneous green fluorescence (low
DCmit) also in both motile and immotile sperm.
Finally, merocyanine-positive cells were stained mostly
in the head (apical portion) and midpiece regions
(Fig. 1), and we could easily distinguish both motile and
immotile merocyanine positive (M540+) and negative
(M540–) sperm.
3.1. Seasonal variations
Sperm concentration declined throughout the four
time periods considered (from September to July),
whereas semen quality, evaluated in terms of sperm
motility, morphology, mitochondrial membrane poten-
tial, and membrane stability, increased from winter to
summer (Table 2).
Poor sperm quality was observed in ejaculates
obtained between September and December, and several
differences were observed between the different time-
Table 3
Seminal characteristicsa from Group A and Group B at the beginning (Januar
to July) of the breeding season.
Parameter January to mid-March M
Group A Group B G
Sperm concentration (�106) 221.45 � 125.99y 139.23 � 47.45z 2
Sperm progressive motility (%)b 32.41 � 15.97y 22.68 � 10.15z 4
Sperm vitality, IP/SYBR14 (%)c 60.58 � 17.46 59.78 � 15.62 7
Morphologically abnormal spermd
Head defects (%) 12.36 � 13.91y 30.50 � 19.52z 8
Midpiece defects (%) 7.74 � 6.33 7.53 � 4.07 5
Tail defects (%) 8.00 � 7.26 9.87 � 7.47 5
Semen pH 7.55 � 0.36 7.63 � 0.36 7
Intact acrosomes (%)e 72.21 � 11.38 69.56 � 11.79 7
Total sperm "DCmit (%)f 65.20 � 16.85y 57.13 � 15.20z 7
Total sperm #DCmit (%)f 29.30 � 15.04 33.23 � 14.82 1
Motile sperm "DCmit (%)f 41.33 � 19.47y 35.87 � 15.75z 5
Immotile sperm "DCmit (%)f 20.81 � 15.28 20.51 � 14.80 1
Motile sperm #DCmit (%)f 16.14 � 13.31 18.91 � 15.61 1
Immotile sperm #DCmit (%)f 15.03 � 11.03 13.78 � 7.59 1
No DCmit motile sperm (%)f 6.72 � 7.73y 10.92 � 7.20z 0
Motile sperm M540+ (%)g 1.04 � 2.10 1.17 � 2.83 0
Immotile sperm M540+ (%)g 35.07 � 21.63 39.77 � 19.51 2
Motile sperm M540– (%)g 48.50 � 22.54 45.08 � 21.88 5
Immotile sperm M540– (%)g 12.38 � 8.36 13.99 � 10.37 1
Total sperm M540+ (%)g 39.11 � 21.42 40.94 � 19.48 2
The semen samples were examined immediately after collection.y,zFor each time period considered, different symbols denote differences (P
a n = 139 semen samples.b Progressive motility sperm observed after collection.c Viable (PI/SYBR14 stained) cells; for each ejaculate, counts were perfd Morphologically abnormal sperm; for each ejaculate, counts were perfe Acrosome intact (PSA-FITC stained) sperm cells; for each ejaculate, cf Sperm cells stained with JC-1; for each ejaculate, counts were performg Sperm cells stained with M540; for each ejaculate, counts were perform
periods considered (Table 2). At the middle of the
breeding season, sperm samples show, in general, better
sperm characteristics; in fact, from the end of March to
mid-May, semen samples were characterized by better
values of acrosome integrity, more sperm with higher
mitochondrial membrane potential, and a lower percen-
tage of merocyanine-stained cells (Table 2 and Table 3).
When simultaneously considering motility and mero-
cyanine labeling, we found that spermatozoa had higher
membrane disorder associated with sperm immobility
(39.88% immotile sperm M540+) in the winter, whereas
in the breeding season the majority of spermatozoa
presented lipid order associated with motility (44.06%
motile sperm M540–). In terms of mitochondrial activity
in progressive motile sperm, in the winter, mean values
for percentage of motile sperm with high IMM (22.17%)
were similar to mean values for percentage of motile
sperm with low IMM (21.98%) (Table 2).
Strong correlations between the several parameters
monitored were found for winter and for the breeding
y to mid-March), middle (mid-March to mid-May), and end (mid-May
id-March to mid-May Mid-May to July
roup A Group B Group A Group B
41.26 � 111.44y 128.77 � 46.10z 190.82 � 84.83 148 � 42.03
5.56 � 12.27y 24.80 � 12.98z 27.94 � 13.81 19.56 � 8.11
3.21 � 15.82y 54.50 � 16.33z 57.62 � 18.00 47.12 � 12.44
.30 � 9.96 16.08 � 14.00 12.50 � 9.67 9.20 � 3.05
.80 � 2.63 6.69 � 4.15 6.93 � 5.45 6.27 � 4.04
.24 � 3.75 7.92 � 4.77 3.40 � 2.91 6.07 � 4.30
.46 � 0.35 7.58 � 0.47 7.29 � 0.30 7.47 � 0.33
9.85 � 9.60 81.93 � 6.75 90.11 � 4.84 86.19 � 9.53
5.82 � 6.90 68.98 � 14.06 74.95 � 8.34 69.71 � 9. 22
9.44 � 8.77 25.27 � 12.79 23.81 � 8.21 30.18 � 8.98
6.32 � 14.54y 40.74 � 18.36z
7.88 � 12.88 25.54 � 18.49
2.63 � 8.18 11.71 � 5.43
2.44 � 6.40 16.51 � 12.59
.77 � 1.23y 5.55 � 4.26z
.82 � 1.12y 3.65 � 5.49z
7.14 � 16.32 34.30 � 12.72
4.83 � 24.53 40.00 � 15.81
4.57 � 15.99 22.25 � 9.91
8.33 � 16.56 38.95 � 13.82
< 0.05) between groups.
ormed on 200 cells.
ormed on 200 cells.
ounts were performed on 200 cells.
ed on 200 cells.
ed on 200 cells.
S. Gamboa et al. / Theriogenology 73 (2010) 950–958 955
Table 4
Pearson correlations between seminal quality parametersa monitored from September to December (rW) and from January to July (rB).
Parameter Morphologically
normal sperm
Motile sperm
"DCmit
Immotile sperm
"DCmit
Immotile sperm
#DCmit
No DCmit
motile sperm
Motile sperm
M540–
Sperm vitalityb rB = 0.45*** rB = 0.30* rW = 0.67*
Motile sperm "DCmitc rW = 0.49** rW = 0.62*
rB = 0.49***
Sperm progressive motilityd rW = 0.51 ** rW = 0.66*
rB = 0.39**
Sperm tail defectse rB = 0.31*
Sperm midpiece defectse rW = 0.54* rB = 0.43***
Total sperm M540+f rB = 0.39**
The semen samples were examined immediately after collection. *P<0.05; **P<0.01; ***P<0.001.a n = 168 semen samples.b Viable (PI/SYBR14 stained) cells; for each ejaculate, counts were performed on 200 cells.c Sperm cells stained with JC-1; for each ejaculate, counts were performed on 200 cells.d Progressive motility sperm observed after collection.e Morphologically abnormal sperm; for each ejaculate, counts were performed on 200 cells.f Sperm cells stained with M540; for each ejaculate, counts were performed on 200 cells.
Table 5
Effect of time period, animal, and fertility on sperm characteristicsa based on MANOVA.
P value
Parameter Interaction Time period
by animal by fertility
Time period Animal Fertility
Sperm concentration (�106) 0.000 0.018 0.000 0.816
Sperm progressive motility (%)b 0.313 0.074 0.000 0.131
Sperm vitality, IP/SYBR14 (%)c 0.133 0.002 0.000 0.027
Morphologically abnormal sperm (%)d 0.001 0.000 0.000 0.143
Head defects (%) 0.003 0.002 0.000 0.024
Midpiece defects (%) 0.709 0.207 0.000 0.901
Tail defects (%) 0.001 0.000 0.000 0.845
Semen pH 0.713 0.021 0.027 0.196
Intact acrosomes (%)e 0.003 0.000 0.000 0.005
Total sperm "DCmit (%)f 0.606 0.000 0.318 0.763
Total sperm #DCmit (%)f 0.409 0.000 0.450 0.706
Motile sperm "DCmit (%)f 0.454 0.001 0.178 0.761
Immotile sperm "DCmit (%)f 0.454 0.432 0.000 0.405
Motile sperm #DCmit (%)f 0.670 0.021 0.008 0.999
Immotile sperm #DCmit (%)f 0.663 0.137 0.068 0.703
No DCmit motile sperm (%)f 0.688 0.022 0.509 0.960
Motile sperm M540+ (%)g 0.594 0.007 0.861 0.940
Immotile sperm M540+ (%)g 0.298 0.122 0.004 0.432
Motile sperm M540– (%)g 0.597 0.011 0.178 0.920
Immotile sperm M540– (%)g 0.188 0.068 0.785 0.481
Total sperm M540+ (%)g 0.132 0.038 0.007 0.432
The semen samples were examined immediately after collection.a n = 168 semen samples.b Progressive motility sperm observed after collection.c Viable (PI/SYBR14 stained) cells; for each ejaculate, counts were performed on 200 cells.d Morphologicaly abnormal sperm; for each ejaculate, counts were performed on 200 cells.e Acrosome intact (PSA-FITC stained) sperm cells; for each ejaculate, counts were performed on 200 cells.f Sperm cells stained with JC-1; for each ejaculate, counts were performed on 200 cells.g Sperm cells stained with M540; for each ejaculate, counts were performed on 200 cells.
S. Gamboa et al. / Theriogenology 73 (2010) 950–958956
season, suggesting some seasonal effects (Table 4). In
general, sperm motility and acrosomal integrity
correlated with motile sperm with both high mitochon-
drial membrane potential and with membrane stability
in motile sperm (r = 0.45, P < 0.001 and r = 0.40,
P < 0.01 for sperm motility and r = 0.35, P < 0.01 and
r = 0.46, P < 0.001 for acrosomal integrity); motile
sperm with high mitochondria membrane potential
correlated with motile sperm presented stable mem-
branes (r = 0.56, P < 0.001).
3.2. Individual variations
Semen quality varied between individual stallions in
the time periods considered. The differences detected
were obvious in more sperm parameters during the
winter and tended to affect less seminal characteristics
with the advancing breeding season (Table 2 and
Table 3).
In winter, stallions with poor fertility showed a major
percentage of immotile spermatozoa with low DCmit.
From January to May, the majority of spermatozoa
(51.71% for Group A; 36.26% for Group B) presented
high DCmit associated with motility (data not shown).
Semen quality was not clearly affected by stallion
fertility at the end of the breeding season, although
interactions between month period, animal, and fertility
were evident (P < 0.05) for sperm concentration, head
and tail sperm anomalies, and acrosomal integrity
(Table 5).
4. Discussion
It has previously been demonstrated that semen
quality in stallions shows variations according to
seasons [5,17] despite some conflicting results obtained
by various researchers [18–20]. Clear significant
seasonal differences in sperm concentration, motility,
viability, sperm morphology, acrosome integrity, and
IMM were evident in this study. Regarding sperm
motility, morphology, and acrosomal integrity, our
results showed no significant differences in semen
collected from September to March. On the other hand,
ejaculates obtained during the winter differed signifi-
cantly from ejaculates obtained at the beginning of the
breeding season (January to mid-March) for sperm
viability, mitochondrial membrane potential, and
membrane stability. A low number of spermatozoa/
mL and the lowest percentage of progressive motile
sperm were registered for the period May to July. This
data contrasts with results from Magistrini et al. [5] and
Hoffmann and Landeck [6] but is in accordance with
those of Janett et al. [19] except for sperm morphologic
characteristics.
It has long been advocated that, besides sperm
concentration, motility, and morphology, other sperm
characteristics and functional probes should be intro-
duced to improve the predictability of routine semen
analysis in various species. We report here that the
introduction of fluorescence-based assays for mem-
brane stability and mitochondrial activity revealed
significant differences for ejaculates collected from
winter to late May. In stallions, fertility may be
maintained at a reduced level outside the breeding
season [7], and, despite reproductive activity not being a
direct function of day length, it is affected by the
endogenous rhythm synchronized by photoperiod (for a
review, see [1]). The techniques used in this study were
able to monitor what could be physiologically relevant
changes due to the circannual rhythm at which
photoperiodic stimuli are received and thus can serve
as markers to detect more functional sperm or possibly a
decrease in function after different treatments or
protocols for semen handling. A shift to high
mitochondrial potential, intact acrosomes, and mem-
brane stability was evident in the breeding season, thus
suggesting that a certain degree of membrane stability
and mitochondrial activity are both relevant for
fertilization.
Stallion fertility depends on a bulk of factors, the
most relevant being the initial quality of the ejaculate. It
is well established that analysis of a given sperm sample
defines the characteristics of that particular sample, not
necessarily its fertilizing ability or, more generally, even
the overall seminal characteristics of the male in
question. This is important when only one or two
samples are obtained for analysis or use, which is
regularly the case, for instance for stallion Studbook
registration. In our work, results from the repeated
collections from the same animal stressed both inter-
stallion and intra-stallion variations in seminal para-
meters. Within-animal and between-animal variation in
sperm traits have been reported by some authors in a
variety of species [21,22] including equines [23–25].
Although a possible breed effect cannot be excluded, in
a previous study of only Puro Sangue Lusitano stallions
[26], a highly significant inter-stallion variation was
also evident, a finding confirmed here. Overall, the
available data clearly indicate that sperm traits vary
within a male over time.
Our results show some fertility group-specific
associations between mitochondrial membrane poten-
tial and sperm function, namely motility, as has also
been described in humans [27], although only in Group
S. Gamboa et al. / Theriogenology 73 (2010) 950–958 957
A was this association related to high mitochondrial
potential. Additionally, stallions with higher fertility
differ from less successful animals in terms of sperm
motility, viability, morphologically abnormal sperm,
and IMM potential during winter and at the beginning
and middle of the breeding season. No differences were
found when sperm was evaluated at the end of the
breeding season.
Jasko et al. [28] and Love et al. [29] found a
relationship between the percentage of spermatozoa
with major defects and fertility in stallions. Our data
stressed that stallions with fertility problems had a
higher percentage of spermatozoa with instable
membranes, and this characteristic was highly corre-
lated with sperm morphologic alterations. Moreover, in
vivo results showed that the best fertility results were
obtained from stallions with a better percentage of live
and acrosome intact cells with high mitochondrial
membrane potential. Reproductive indexes confirmed
that poor semen quality stallions had the worst
pregnancy rates.
In conclusion, seminal parameters monitored are
affected mostly by time period, followed by animal and
lastly by fertility, stressing the importance of individual
variations in out-bred animal models.
Acknowledgments
Sandra Gamboa wishes to thank Nobre de Oliveira
and all the staff of the Animal Reproduction Laboratory
at ESAC. Sandra Gamboa is the recipient of a PhD
scholarship (SFRH/BD/37612/2007) from the Portu-
guese Foundation for Science and Technology, Portu-
gal. This work was supported by the Agricultural
School of Coimbra and by Fundacao para a Ciencia e
Tecnologia (POCTI/CVT/49102/2002), Portugal.
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