Animal Reproduction Science 156 (2015) 128–134

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Animal Reproduction Science

journal homepage: www.elsevier.com/locate/anireprosci

Sperm population structure in high and low field

fertility rams
J.L. Yániz ∗ , I. Palacín, S. Vicente-Fiel, J.A. Sánchez-Nadal, P. Santolaria
TECNOGAM Research Group, Environmental Sciences Institute (IUCA), University of Zaragoza, Huesca, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to investigate whether differences in field fertility of
Received 29 November 2014 rams are reflected in differences in sperm morphometric and kinematic population struc-
Received in revised form 7 February 2015 tures. The association between sperm morphometric and kinematic subpopulations was
Accepted 11 March 2015
also investigated. Ejaculates from 8 adult rams, 4 with high and 4 with low field fertility,
Available online 21 March 2015
were collected weekly using an artificial vagina over 6 consecutive weeks. Analyses of sperm
motility using computer-assisted sperm analysis (CASA) and sperm nuclear morphome-
Keywords:
try using computer-assisted sperm morphometry-fluorescence were performed. Clustering
Sheep
procedures using the kinematic and morphometric data from high and low field fertility
Sperm subpopulations
CASA rams resulted in the classification of spermatozoa in three kinematic and three morphomet-
CASMA ric sperm subpopulations. The distribution of subpopulations between rams of high and low
field fertility was significantly different (P < 0.05), with higher percentages of spermatozoa
exhibiting fast and linear movements and those with large and long nuclei in the high fertil-
ity group. However, these subpopulations were not correlated. Logistic regression analyses
were also performed to evaluate the relative utility of sperm subpopulations to classify
rams in high and low field fertility. Total progressive sperm motility and the proportion of
large and long spermatozoa were identified as the most consistent indicators of fertility. It
was concluded that high and low fertility rams had clear differences in morphometric and
kinematic sperm subpopulations, and that the most consistent indicators of fertility were
the total progressive motility and the proportion of spermatozoa with large and long head
present in the ejaculate.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction revealed wide ranges of variation and showed low pre-

The predictive capacity of sperm quality analysis on
potential fertility of males and ejaculates needs to be
improved. Although correlations have been described
between several sperm quality parameters and fertility,
most of these correlations, albeit statistically significant,
∗ Corresponding author at: Escuela Politécnica Superior de Huesca, Uni-
versidad de Zaragoza, Ctra. Cuarte S/N, 22071 Huesca, Spain.
Tel.: +34 974 239312; fax: +34 974239302.
E-mail address: jyaniz@unizar.es (J.L. Yániz).
dictive value (Rodriguez-Martinez, 2003). The incomplete
information provided by the analyses lie behind this
restricted ability to achieve accurate predictions. In this
sense, although the existence of sperm subpopulations in
the ejaculate is widely recognised (Holt and Van Look,
2004), the majority of studies have addressed the rela-
tionship between sperm traits and male fertility based on
average values of sperm parameters.
Interest in sperm subpopulations comes from the
increasing evidence that the heterogeneity among them
may have functional relevance. For example, relations
have been found between the ejaculate heterogeneity and

http://dx.doi.org/10.1016/j.anireprosci.2015.03.012
0378-4320/© 2015 Elsevier B.V. All rights reserved.
 J.L. Yániz et al. / Animal Reproduction Science 156 (2015) 128–134
fertility (de Paz et al., 2011; Ramon et al., 2013), or
the ability to survive cryopreservation (Thurston et al.,
2001; Nunez Martinez et al., 2006; Ortega-Ferrusola et al.,
2009). Theoretically, a greater heterogeneity of spermato-
zoa would ensure greater potential to fertilise an oocyte at
some unpredictable interval after ejaculation (Curry, 2000).
Identification of sperm subpopulations sharing com-
mon characteristics has been conducted based on cluster-
ing procedures on the kinematic or morphometric data
provided by computer-assisted sperm analysis CASA sys-
tems (Martinez-Pastor et al., 2005; Nunez Martinez et al.,
2006). The possible association between sperm morphol-
ogy and sperm velocity has been suggested in several
interspecific studies (Malo et al., 2006; Gomendio and
Roldan, 2008; Tourmente et al., 2011), while other study
disagrees with these findings (Humphries et al., 2008). In
the only intraspecific study evaluating this aspect, Ramon
et al., 2013 described that sperm morphometric and kine-
matic subpopulations were strongly interrelated in the
Iberian red deer, and found an association between these
subpopulations and field fertility.
In the ram, some studies have described the existence
of morphometric (de Paz et al., 2011; Martí et al., 2011;
Maroto-Morales et al., 2012; Vicente-Fiel et al., 2013) and
kinematic (Garcia-Alvarez et al., 2014) sperm subpopula-
tions. However, to our knowledge, there is only one study
evaluating the possible association between sperm mor-
phometric subpopulations and fertility (de Paz et al., 2011)
and no studies have evaluated this aspect in relation to
the sperm kinematic subpopulations in ovine species. In
a recent study, we described a relationship between sev-
eral sperm quality parameters, using average values, and
ram field fertility (Vicente-Fiel et al., 2014). The aim of the
present study was to investigate whether differences in
field fertility of rams are related to differences in sperm
morphometric and kinematic population structure.
2. Materials and methods
2.1. Reagents
Unless otherwise stated, all chemicals used were
obtained from Sigma–Aldrich Chemical Company
(Alcobendas, Madrid, Spain).
2.2. Animals and semen processing
All animal procedures were performed in accor-
dance with the Spanish Animal Protection Regulation
RD223/1988, which conforms to European Union Regu-
lation 86/609. Rams used in the study belonged to the
Rasa Aragonesa breed selection scheme followed by UPRA-
Grupo Pastores. All the animals were fed a standard diet
with water offered ad-libitum. Semen was collected weekly
with the aid of an artificial vagina from 8 adult Rasa
Aragonesa rams, 4 with high and 4 with low field fertility,
over 6 consecutive weeks. A routine of two semen col-
lections per week was established. Only one ejaculate per
male per week was used in the 6 wk of the study and the
other was discarded. The rams were classified as having
high and low fertility based on data of a previous large field
129
epidemiological study (Palacín et al., 2012), were rams with
positive and negative impact on fertility were detected. The
samples used in the current study were collected immedi-
ately after the end of the study of Palacín et al. (2012) so
that possible variations in ram fertility were minimised. To
further validate this classification of rams, a new epidemi-
ological analysis was conducted including also the data
between the end of the study of Palacín et al. (2012) and
the end of sample collection period in the present study.
The results obtained confirmed that the rams were cor-
rectly classified in the high and low fertility groups (data
not shown).
Sperm motility and concentration were measured using
a computer-assisted sperm analyser (CASA) (ISAS® , Ver-
sion 1.0; PROISER, Paterna, Spain) after placing a diluted
semen sample in a pre-warmed Makler chamber or in a
Burker chamber in duplicate, respectively. The semen of
each ejaculate was pre-diluted in INRA-96® diluent (IMV
Technologies, L’Aigle, France) to 200 × 106 sperm/mL, kept
in sterile glass tubes and stored at 37 ◦ C until laboratory
analysis was performed within 3 h of sample collection.
Semen samples were carefully mixed, and sample aliquots
were diluted to 50 × 106 sperm/mL using INRA96® for
sperm motility assessment (Vicente-Fiel et al., 2014), or
prepared for sperm morphometry assessment as previ-
ously described (Yániz et al., 2012). Briefly, semen smears
were allowed to air dry for a minimum of 2 h, fixed with 2%
glutaraldehyde in PBS for a 3-min exposure, washed thor-
oughly in distilled water and labelled with Hoechst 33342
as detailed below.
2.3. Sperm motility determination by CASA
A computer-assisted sperm analyser (ISAS® , Version
1.0, PROISER, Valencia, Spain) and an Olympus BX40 micro-
scope (Olympus Optical Co., Tokio, Japan) equipped with
a negative phase-contrast 10× objective and heated stage
set at 37 ◦ C, were used to assess sperm motility. Sample
aliquots (5 ␮L) per duplicate were placed in a pre-warmed
slide and immediately covered with 22 × 22 mm glass
coverslips (Palacín et al., 2013). The motility variables mea-
sured included the sperm cell motility percentage (MS,
%), the progressive motility percentage (PS, %), curvilin-
ear velocity (VCL, ␮m/s), straight line velocity (VSL, ␮m/s),
average path velocity (VAP, ␮m/s), sperm linearity (LIN,
as a measure of a curvilinear path, VSL/VCL), straightness
(STR, as the linearity of the average path, VSL/VAP), wob-
ble (WOB, oscillation measure of the actual path about the
average path, VAP/VCL), amplitude of lateral sperm head
displacement (ALH, ␮m) and beat cross frequency (BCF, Hz)
(Palacín et al., 2013).
2.4. Fluorescence imaging and computer-assisted sperm
morphometry analyses (CASMA-F)
Semen smears were stained by placing 20 ␮L of a
Hoechst 33342 suspension (20 ␮g/mL in a TRIS-based solu-
tion) between the slide and a coverslip, which was then
incubated for 20 min in the dark at room temperature
(Yániz et al., 2012). The coverslip was then removed and
the slide was washed thoroughly with distilled water and
130 J.L. Yániz et al. / Animal Reproduction Science 156 (2015) 128–134

allowed to dry. Digital images of the fluorescence-labelled Table 1

Results of the principal component analysis (PCA) performed on the
sperm nuclei were recorded using a setup composed of
computer-assisted sperm analysis (CASA) sperm motility data in the ram
an epifluorescence microscope (DM4500B, Leica, Wetzlar, (n = 48).
Germany; A-UV filter cube, BP340-380 excitation filter,
LP425 suppressor filter, dichromatic mirror: DM400) with Motility parameters PC1 PC2

a 63X plan apochromatic objective, and photographed with VCL 0.589 0.797
a Canon Eos 400D digital camera (Canon Inc., Tokyo, Japan). VSL 0.963 0.195
VAP 0.867 0.430
The camera was controlled by a computer using DSLR
LIN 0.932 −0.296
Remote Pro software (Breeze Systems, Camberley, UK). STR 0.770 −0.251
From each captured image, sperm nuclei morphome- WOB 0.879 −0.250
try was automatically analysed using the Image J software, ALH −0.417 0.819
with a plug-in created for this purpose (Yániz et al., PC: principal component; VCL: curvilinear velocity; VSL: straight line
2012). Each sperm nucleus was measured for four primary velocity; VAP: average path velocity; LIN: linearity; SRT: straightness;
parameters and four derived parameters for nuclear shape. WOB: wobble; ALH: amplitude of lateral sperm head displacement.

Primary parameters were Area (A, in ␮2 , as the sum of all

pixel areas contained within the boundary), Perimeter (P, in
␮, as the sum of external boundaries), Length (L) and Width
(W) (in ␮, the highest and lowest values, respectively, of
the Feret diameters, i.e., the projection of the sperm head
on the horizontal axis measured at angles of rotation of
0◦ , 30◦ , 60◦ , 90◦ , 120◦ and 150◦ ; Length and Width are not
necessarily orthogonal)] Derived nuclear shape parame-
ters were Ellipticity (L/W), Rugosity (4␲A/P2), Elongation
((L − W)/(L + W)) and Regularity (␲LW/4A).
2.5. Statistical analysis
The values obtained were expressed as mean ± standard
error of the mean (SEM). Statistical analyses were per-
formed using the SPSS package, version 15.0 (SPSS Inc.,
Chicago, IL, USA). The statistical level of significance was set
at P < 0.05. Clustering procedures were further performed
to identify sperm subpopulations using the kinematic or
morphometric data (Martinez-Pastor et al., 2005; Nunez
Martinez et al., 2006; Vicente-Fiel et al., 2013). The first
step was to perform a principal component analysis (PCA)
of the morphometry data. The purpose of PCA is to derive
a small number of linear combinations (principal com-
ponents) from a set of variables that retain as much of
the information in the original variables as possible. This
allows the summarising of many variables in a few, jointly
uncorrelated, principal components. A good result is when
we obtain a few principal components accounting for a
high proportion of the total variance. To select the number
of principal components that should be used in the next
step of our analysis, we followed the criterion of select-
ing only those with an eigenvalue (variance extracted for
that particular principal component) higher than 1 (Kaiser
criterion). The second step was to perform a 2-step clus-
ter procedure with the sperm-derived indexes obtained
after the PCA. This analysis allowed the identification of
sperm subpopulations and the detection of the outliers.
After characterising sperm subpopulations, we performed
a two-way correlation analysis including all sperm sub-
populations to explore possible associations between both
sperm nucleus morphometry and sperm velocity. Analysis
of variance (ANOVA) was used to explore the relationships
between the proportion of each sperm subpopulation in
the sperm sample and fertility. The statistical level of sig-
nificance was set at P < 0.05.
Finally, logistic regression analyses were performed to
explore the importance of the morphometric/kinematic
subpopulation size and the total/progressive motility per-
centage of each analysis for predicting ram fertility. Total
and progressive motility percentages were included in the
logistic regression analysis because these CASA variables
were not included in the multivariate cluster analysis.
Logistic regression analysis (SPSS software, Logistic pro-
cedure) was conducted according to the Hosmer and
Lemeshow method (Hosmer and Lemeshow, 1989). This
method involves five steps as follows: (i) preliminary
screening of all variables for univariate associations; (ii)
construction of a full model using all the variables found
to be significant in the univariate analysis; (iii) stepwise
removal of non-significant variables from the full model
and comparison of the reduced model with the previous
model for model fit and confounding; (iv) evaluation of
interactions among variables and (v) assessment of model
fit using Hosmer–Lemeshow statistics. Variables with uni-
variable associations showing P < 0.25 were included in the
initial model. Modelling was continued until all the main
effects or interaction terms were significant according to
the likelihood-ratio at P < 0.05.
3. Results
In the analysis of kinematic variables, PCA rendered
two principal components with eigenvalues over 1 (PC1
and PC2; Table 1), which accounted for more than 88%
of the cumulative variance. The first principal component
was related to rapid and linear movement, whereas the
second principal component was related to rapid erratic
movement, including wide head lateral displacement.
The two-step clustering analyses, using the two principal
Table 2
Results of the 2-step cluster procedure in the ram (n = 48) with the kine-
matic indexes (PC) as variables.
Subpopulation PC 1 PC 2
Mean SEM Mean SEM
SP1mot −1.02 0.006 −1.42 0.004
SP2mot 0.68 0.002 −0.38 0.002
SP3mot −0.92 0.004 1.04 0.004
Outliers cluster −2.43 0.104 4.13 0.110
PC: principal component; SPmot : motility subpopulation
 J.L. Yániz et al. / Animal Reproduction Science 156 (2015) 128–134 131

Table 3
Kinematic descriptors (mean ± SEM) of sperm for each cluster (subpopulations) found after the PCA-Cluster analysis in the ram (n = 48).

Kinematic descriptors Sperm subpopulations

SP1mot SP2mot SP3mot

VCL 58.06 ± 0.0194a 147.23 ± 0.089b 145.46 ± 0.153b

VSL 36.45 ± 0.186a 129.33 ± 0.099b 60.36 ± 0.162a
VAP 43.50 ± 0.180a 136.59 ± 0.097b 93.92 ± 0.178c
LIN 0.56 ± 0.002a 0.88 ± 0.000b 0.40 ± 0.001a
STR 0.75 ± 0.002a 0.94 ± 0,000b 0.65 ± 0.001a
WOB 0.72 ± 0.001a 0.92 ± 0.000b 0.63 ± 0.001a
ALH 2.13 ± 0.006a 2.79 ± 0.004a 5.25 ± 0.007b

SPmot : motility subpopulation; VCL: curvilinear velocity; VSL: straight line velocity; VAP: average path velocity; LIN: linearity; SRT: straightness; WOB:
wobble; ALH: amplitude of lateral sperm head displacement.
a–c
Denote differences within rows at P < 0.0001.

Table 4 is defined mainly by primary (A, P and W), the second

Results of the principal component analysis (PCA) performed on the
(PC2) by primary (L) and secondary factors (Ellipticity and
computer-assisted sperm morphometry-fluorescence analysis (CASMA-
F) data in the ram (n = 48).
Elongation) and the third (PC3) by Regularity. The sec-
ond clustering analysis revealed the existence of 3 sperm
Morphometric parameters PC1 PC2 PC3 subpopulations (Table 5). Subpopulation 1 (SP1morpho )
Area 0.91 0.38 −0.17 included small spermatozoa (low A, P, L, W). Subpopu-
Perimeter 0.70 0.69 0.09 lation 2 (SP2morpho ) consisted of average size and round
Length 0.52 0.84 −0.07
spermatozoa (low L, Ellipticity and Elongation). Subpopu-
Width 0.99 −0.05 0.15
Ellipticity −0.60 0.77 −0.18 lation 3 (SP3morpho ) included large and long spermatozoa
Rugosity 0.49 −0.60 −0.49 (high A, L). Table 6 shows the mean morphometric values
Elongation −0.60 0.76 −0.21 for each subpopulation. Of total spermatozoa, 17.8, 34.8
Regularity −0.01 0.07 0.95 and 47.0% were included in subpopulations 1, 2 and 3,
PC: principal component. respectively.
The distribution of subpopulations between rams of
high and low field fertility was significantly different, with a
components as variables, revealed the presence of three higher proportion of spermatozoa of the SP2mot (rapid and
sperm subpopulations in rams (Table 2). Subpopulation linear) and of the SP3morpho (large and long spermatozoa),
1 (SP1mot ) has negative values for PC1 and PC2, so this and a lower proportion of the SP3mot (rapid nonlinear) in
cluster would comprise slow, nonlinear spermatozoa with the higher than in the lower fertility rams (P < 0.05, Table 7).
low ALH. Subpopulation 2 (SP2mot ) has positive values for We searched for possible correlations between morphom-
PC1 and negative for PC2, so this cluster would include etry and velocity subpopulations (percentages of each) to
rapid and linear spermatozoa. In the same sense, sub- assess if spermatozoa in a particular morphometric sperm
population 3 (SP3mot ) has negative values for PC1 and subpopulation had a characteristic movement. We found
positive for PC2, so this cluster would include rapid, non- that the proportion of spermatozoa with a small nucleus
linear spermatozoa, with high ALH. These characteristics (SP1morpho ) showed a positive correlation (r = 0.32, P < 0.05)
are reflected in the mean values of the motility descriptors with the proportion of spermatozoa demonstrating a slow,
(Table 3). Of total motile spermatozoa, 17.9%, 53.1% and nonlinear movement with low ALH (SP1mot ) and a nega-
29.0% were included in Subpopulations 1, 2 and 3, respec- tive correlation (r = −0.33, P < 0.05) with the proportion of
tively. spermatozoa demonstrating a rapid and linear movement
In the analysis of nuclear sperm morphometry with (SP2mot ).
CASMA-F technology, PCA analysis revealed 3 components Table 8 shows the odds ratios of variables finally
with eigenvalues over 1, representing more than 95.4% of included in the logistic model for ram fertility. No sig-
the cumulative variance (Table 4). The first factor (PC1) nificant interactions were found. Based on the odds

Table 5
Results of the 2-step cluster procedure in the ram (n = 48) with the morphometric indexes (PC) as variables.

Subpopulation PC 1 PC 2 PC 3

Means SD Means SD Means SD

SP1morpho −1.09 0.020 0.06 0.018 0.64 0.022

SP2morpho 0.19 0.012 −0.82 0.010 −0.02 0.010
SP3morpho 0.40 0.015 0.72 0.011 −0.41 0.011
Outliers cluster 1.49 0.548 3.58 0.806 8.58 0.934

PC: principal component; SPmorpho : morphometric subpopulation

132 J.L. Yániz et al. / Animal Reproduction Science 156 (2015) 128–134

Table 6
Morphometric descriptors of sperm nucleus (mean ±SEM) for each cluster (subpopulations) found after the PCA-Cluster analysis in the ram (n = 48).

Morphometric descriptors Sperm subpopulations

SP1morpho SP2morpho SP3morpho

Area (␮m2 ) 27.92 ± 0.035a 29.65 ± 0.022b 31.28 ± 0.024c

Perimeter (␮m) 21.69 ± 0.014a 21.87 ± 0.008b 22.67 ± 0.008c
Length (␮m) 8.05 ± 0.005a 8.04 ± 0.003a 8.42 ± 0.003b
Width (␮m) 4.42 ± 0.004a 4.65 ± 0.002b 4.66 ± 0.003b
Ellipticity (%) 1.82 ± 0.001a 1.73 ± 0.001b 1.80 ± 0.001a
Rugosity 0.75 ± 0.001a 0.78 ± 0.000b 0.76 ± 0.000c
Elongation 0.29 ± 0.000a 0.27 ± 0.000b 0.29 ± 0.000a
Regularity 1.00 ± 0.000a 0.99 ± 0.000b 0.99 ± 0.000b

SPmorpho : morphometric subpopulation.

a–c
Denote differences within rows at P < 0.0001.

Table 7 subpopulations and ram fertility. The evaluation of male

Differences in the relative size of each sperm subpopulations (cluster, per-
fertility by test inseminations in the field is expensive
centage of sperm) between high (n = 24) and low (n = 24) fertility groups
rams defined with the CASA motility or CASMA-F morphometry analytical
and time-consuming. Consequently, there is a continuous
procedures. Data are represented as Mean ± S.E.M. search a need for an in vitro test with a higher predic-
tion capacity of the potential fertility of males (Vicente-Fiel
Sperm cluster Group Size of the cluster (%)
et al., 2014). The development of predictive in vitro testing
CASA of male field fertility requires an understanding of the basis
SP1mot High 18.00 ± 2.34a
of the differences in field fertility between individual males.
Low 21.49 ± 2.34a
Given that the ejaculate has a heterogeneous population
SP2mot High 61.05 ± 3.42c of spermatozoa, and that the distribution of sperm sub-
Low 46.16 ± 3.42d
populations varies between males, the present study was
SP3mot High 20.95 ± 2.16e directed to investigate the possible associations between
Low 32.26 ± 2.16f
sperm subpopulations and field fertility. The association
between sperm morphometry and motility subpopulations
CASMA-F
SP1morpho High 17.75 ± 2.93a was also investigated for the first time in rams. The three
Low 25.57 ± 2.93a kinematic sperm subpopulations described in the present
SP2morpho High 34.77 ± 4.02a
study are similar to those described in a previous study
Low 41.71 ± 4.02a in the Manchega ovine breed (Garcia-Alvarez et al., 2014).
However, to our knowledge, this is the first study where
SP3morpho High 47.00 ± 4.50a
Low 32.48 ± 4.50b the possible relations between kinematic sperm subpopu-
lations and fertility have been investigated in ovine species.
CASA: computer-assisted sperm analysis; CASMA-F: computer-assisted
sperm morphometry-fluorescence analysis; SPmot : motility subpopula-
The three morphometric sperm subpopulations identi-
tion; SPmorpho : morphometric subpopulation. fied in rams in the present study were small, round and
a–b
Superscripts denote differences between high and low fertility group. large-long. These subpopulations are in agreement with
a–b
P < 0.05. those described in a recent study, where we identified three
c–d
P < 0.01.
e–f morphometric sperm subpopulations using the CASMA-F
P = 0.001.
method: large, average size-round and small (Vicente-Fiel
et al., 2013). However, other studies in ovine species have
ratio, the likelihood of high fertility increased in semen described the existence of 3–4 sperm subpopulations, dif-
samples with high total progressive motility (by a fac- ferentiated according to the head Area, Length, Ellipticity
tor of 1.18) and in those with a higher proportion of and Elongation (Martí et al., 2011), or four sperm subpopu-
morphometric subpopulation 3 (large and long spermato- lations differentiated by head sperm length and p2a (the
zoa, by a factor of 1.05). inverse of Rugosity described in this study) parameters
(Maroto-Morales et al., 2012). Differences between stud-
4. Discussion ies may be attributed to the different breeds and methods
used.
This study provides new insight regarding the asso- Our results show that rams with high and low field fer-
ciation between sperm morphometric and kinematic tility show differences in the distribution of kinematic and

Table 8
Odds ratios of the variables included in the final logistic regression model for ram fertility.

Factor n Odds ratio 95% Confidence interval P

Progressive sperm motility (%) 48 1.18 1.07–1.29 <0.001

SP3morpho (%) 48 1.05 1.00–1.09 <0.05

SPmorpho : morphometric subpopulation. Likelihood ratio test = 35537; 2df, P < 0.0001.
Hosmer and Lemeshow Goodness-of-fit test = 5.50; 8 df, P = 0.7 (the model fits).
 J.L. Yániz et al. / Animal Reproduction Science 156 (2015) 128–134
morphometric sperm subpopulations. Rams with superior
field fertility displayed increased proportion of spermato-
zoa with efficient swimming (fast and linear) and with
large and long heads. Both sperm subpopulations might be
related to a higher sperm migratory efficiency through the
female genital tract. In the ovine, the cervix and the isth-
mus are the main barriers for spermatozoa after cervical
AI (Yániz et al., 2014). In these anatomical structures, the
epithelial mucosa produces viscous mucus that spermato-
zoa must penetrate to reach the fertilisation site in the
ampullary-isthmic junction of the oviduct. In conjunction
with local viscous secretions, the complex surface archi-
tecture of the cervical and oviductal mucosa (Yániz et al.,
2014) may hinder the passage of spermatozoa. Ejaculates
with a higher proportion of spermatozoa with a rapid and
linear movement would probably have a more efficient
migration through the female reproductive tract. In agree-
ment with our results, Ramon et al. (2013) observed that
red deer males with high fertility rates had ejaculates with
high percentages of spermatozoa exhibiting fast and lin-
ear movements. Spermatozoa with large and long heads
might also be related to a more efficient sperm migration. In
sperm competition studies, it has been demonstrated that
all sperm components increase in size simultaneously, and
this might be associated with an improved sperm migra-
tory efficiency (Tourmente et al., 2011).
This is also the first time that the association between
sperm design and swimming velocity has been investigated
in the ram. In the only intraspecific study evaluating this
aspect, a clear correlation between sperm size and velocity
was described in the Iberian red deer (Ramon et al., 2013).
The authors observed that spermatozoa with rapid and lin-
ear movement were strongly correlated with spermatozoa
having a small and elongated head, being both subpopula-
tions more frequent in high-fertility males. In the present
study, however, fast and linear spermatozoa and those hav-
ing large and long nuclei were related to high fertility,
but both subpopulations were not correlated. An asso-
ciation between spermatozoa with a small nucleus and
those having a slow, nonlinear movement was found, but
a clear correspondence between morphometric and kine-
matic sperm subpopulations was not observed in the ram.
The study of the association between sperm subpopu-
lations and ram fertility using logistic regression methods
provides new insight into the relative importance of the
different parameters. Total and progressive sperm motil-
ity percentages were also included in the model as these
CASA variables are complementary to the sperm subpop-
ulations. Total progressive motility and the proportion of
large and long spermatozoa were identified as the most
consistent indicators of fertility. In agreement with our
results, progressive sperm motility has been considered a
good indicator of male fertility in various species (Gadea,
2005; Kasimanickam et al., 2007; Broekhuijse et al., 2012).
5. Conclusions
It was concluded that high and low fertility rams
present clear differences in morphometric and kinematic
sperm subpopulations, and that the most consistent indi-
cators of fertility were the total progressive motility and
133
the proportion of large and long spermatozoa present in
the ejaculate. This may be useful in the selection of males
for artificial insemination based on sperm quality data.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank Neil Macowan for assistance with
the English translation and the CITA (DGA) for their help
with procuring semen samples. This work was supported
by the Spanish MINECO (grants IPT-010000-2010-33 and
AGL2011-30353-C02-01), and the DGA-FSE (grant A40).
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