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.

References

[1] Gerlach T, Aurich JE. Regulation of seasonal reproductive

activity in the stallion, ram and hamster. Anim Reprod Sci

2000;58:197–213.

[2] Chemineau P, Guillaume D, Migaud M, Thiery JC, Pellicer-

Rubio MT, Malpaux B. Seasonality of reproduction in mammals:

intimate regulatory mechanisms and practical implications.

Reprod Domest Anim 2008;43:40–7.

[3] Hughes JP, Stabenfeldt GH, Evans JW. The oestrous cycle of the

mare. J Reprod Fertil Suppl 1975;23:161–6.

[4] Johnson L. Seasonal differences in equine spermatocytogenesis.

Biol Reprod 1991;44:284–91.

[5] Magistrini M, Chanteloube P, Palmer E. Influence of season and

frequency of ejaculation on production of stallion semen for

freezing. J Reprod Fertil Suppl 1987;35:127–33.

[6] Hoffmann B, Landeck A. Testicular endocrine function, season-

ality and semen quality of the stallion. Anim Reprod Sci

1999;57:89–98.

[7] Guillaume D. Photoperiod action on equine reproduction. INRA

Prod Anim 1996;9:61–9.

[8] Skalet LH, Rodrigues HD, Goyal HO, Maloney MA, Vig MM,

Noble RC. Effects of age and season on the type and occurrence of

sperm abnormalities in Nubian bucks. Am J Vet Res 1988;49:1284–

9.

[9] Varner DD. Developments in stallion semen evaluation. Ther-

iogenology 2008;70:448–62.

[10] Silva PFN, Gadella BM. Detection of damage in mammalian

sperm cells. Theriogenology 2006;65:958–78.

[11] Gamboa S, Ramalho-Santos J. SNARE proteins and caveolin-1

in stallion spermatozoa: possible implications for fertility. Ther-

iogenology 2005;64:275–91.

[12] Foote RH. Effect of processing and measuring procedures on

estimated size of bull sperm heads. Theriogenology 2003;59:

1765–73.

[13] Harrison RA, Ashworth PJ, Miller NG. Bicarbonate/CO2, an

effector of capacitation, induces a rapid and reversible change in

the lipid architecture of boar sperm plasma membranes. Mol

Reprod Dev 1996;45:378–91.

[14] Smiley ST, Reers M, Mottola-Hartshorn C, Lin M, Chen A,

Smith TW, et al. Intracellular heterogeneity in mitochondrial

membrane potentials revealed by a J-aggregate-forming lipo-

philic cation JC-1. Proc Natl Acad Sci U S A 1991;88:3671–5.

[15] Reers M, Smith TW, Chen LB. J-aggregate formation of a

carbocyanine as a quantitative fluorescent indicator of mem-

brane potential. Biochemistry 1991;30:4480–6.

[16] Gravance CG, Garner DL, Baumber J, Ball BA. Assessment of

equine sperm mitochondrial function using JC-1. Theriogenol-

ogy 2000;53:1691–703.

[17] Morte MI, Rodrigues AM, Soares D, Rodrigues AS, Gamboa S,

Ramalho-Santos J. The quantification of lipid and protein oxidation

in stallion spermatozoa and seminal plasma: seasonal distinctions

and correlations with DNA strand breaks, classical seminal para-

meters and stallion fertility. Anim Reprod Sci 2008;106:36–47.

[18] Jasko DJ, Lein DH, Foote RH. The repeatability and effect of

season on seminal characteristics and computer-aided sperm

analysis in the stallion. Theriogenology 1991;35:317–27.

[19] Janett F, Thun R, Niedere K, Burger D, Hassig M. Seasonal

changes in semen quality and freezability in the Warmblood

stallion. Theriogenology 2003;60:446–53.

[20] Janett F, Thun R, Bettschen D, Burger D, Hassig M. Seasonal

changes of semen quality and freezability in Franches-Mon-

tagnes stallions. Anim Reprod Sci 2003;77:213–21.

[21] Breed WG, Bauer M, Wade R, Thitipramote N, Suwajarat J,

Yelland L. Intra-individual variation in sperm tail length in

murine rodents. J Zool 2007;272:299–304.

[22] Erenpreiss J, Bungum M, Spano M, Elzanaty S, Orbidans J,

Giwercman A. Intra-individual variation in sperm chromatin

structure assay parameters in men from infertile couples: clinical

implications. Hum Reprod 2006;21:2061–4.

[23] Lopez-Fernandez C, Crespo F, Arroyo F, Fernandez JL, Arana P,

Johnston SD, Gosalvez J. Dynamics of sperm DNA fragmentation

in domestic animals: II. The stallion. Theriogenology 2007;68:

1240–50.

[24] Cortes-Gutierrez EI, Crespo F, Gosalvez A, Davila-Rodrıguez

MI, Lopez-Fernandez C, Gosalvez J. DNA fragmentation in

frozen sperm of Equus asinus: Zamorano-Leones, a breed at risk

of extinction. Theriogenology 2008;69:1022–32.

S. Gamboa et al. / Theriogenology 73 (2010) 950–958958

[25] Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer

E. Equine frozen semen: freezability and fertility field results.

Theriogenology 1997;48:907–17.

[26] Gamboa S, Machado-Faria M, Ramalho-Santos J. Seminal traits,

suitability for semen preservation and fertility in the native

Portuguese horse breeds Puro Sangue Lusitano and Sorraia:

implications for stallion classification and assisted reproduction.

Anim Reprod Sci 2009;13:102–13.

[27] Marchetti C, Obert G, Deffosez A, Formstecher P, Marchetti

P. Study of mitochondrial membrane potential, reactive

oxygen species, DNA fragmentatation and cell viability by

flow cytometry in human sperm. Hum Reprod 2002;17:1257–

65.

[28] Jasko DJ, Lein DH, Foote RH. A comparison of two computer-

automated semen analysis instruments for the evaluation of

sperm motion characteristics in the stallion. J Androl 1990;11:

453–9.

[29] Love CC, Varner DD, Thompson JA. Intra- and inter-stallion

variation in sperm morphology and their relationship with

fertility. J Reprod Fertil 2000;56:93–100.