J. Appl. Ichthyol. 24 (2008), 398–405
2008 Blackwell Verlag, Berlin
ISSN 0175–8659
Received: January 20, 2008
Accepted: May 12, 2008
doi: 10.1111/j.1439-0426.2008.01125.x
Sperm morphology and its influence on swimming speed in Atlantic cod
By V. M. Tuset1,2, E. A. Trippel2 and J. de Monserrat3
1
Departamento de Biologı´a Pesquera, Instituto Canario de Ciencias Marinas, Telde (Las Palmas), Canary Islands, Spain;
Department of Fisheries and Oceans Canada, Biological Station, St Andrews, NB, Canada; 3Unidad de Análisis por Imagen,
Laboratorio de análisis Dr Echevarne, Barcelona, Spain
2
Summary
A protocol for staining fish spermatozoa using Hemacolorstain was developed for light microscopy and successfully
applied to Atlantic cod (Gadus morhua). Sperm head morphology was characterized by size (length, width, area and
perimeter) and shape (ellipticity, rugosity, elongation and
regularity) (n = 6500 spermatozoa), and tail length (n = 260
spermatozoa) of 12 individual cod. Two spermatozoa heads
sperm were clearly identified: round and elongated, being this
last one more abundant (86.3%). No evidence was detected in
tail length for both head types. Tails were 96.4% length of
sperm and no difference in tail length was detected between
head types. A positive correlation existed between head and
tail length, with variability existing among males. Sperm
swimming speeds varied among males with a maximum
curvilinear velocity between 151.5 and 201.5 lm s)1. Mean
swimming speed declined by 8.2% from 30 to 70 s postactivation. Spermatocrit was negatively correlated with curvilinear velocity at 30 s post-activation. Males with short sperm
heads maintained their swimming velocity for longer periods
that those with long heads. FultonÕs condition factor was
negatively correlated with straightness of path.
Introduction
Male gametes and their inherent fertilization capacity is an
aspect of fish reproductive biology that has received broadened
interest in recent years (Trippel, 2003; Rurangwa et al., 2004).
Semen quality as it relates to fertilization is a key component
of mating success and broodstock development (Gage et al.,
2004; Casselman et al., 2006). Quality of milt is defined in
terms of sperm morphology, density, motility, osmolality, pH,
chemical composition, enzymatic activity and ATP concentration (Kime et al., 2001; Rurangwa et al., 2004; Asturiano
et al., 2006). Sperm morphology in relation to physiological
performance, however, has been the focus of only a few
studies, Salmo salar (Gage et al., 2002), Carassius auratus
(Van-Look and Kime, 2003) and Anguilla anguilla (MarcoJiménez et al., 2006).
Sperm morphology has normally been investigated using
transmission or scanning electron microscopy (Jamieson, 1991;
Mattei, 1991; Medina et al., 2003; Lahnsteiner and Mansour,
2004). For light microscopy, spermatozoa are commonly fixed
in glutaraldehyde and viewed by negative-phase contrast
microscopy (Gage et al., 1998, 2002; Balshine et al., 2001;
Vladić et al., 2002; Van-Look and Kime, 2003; Burness et al.,
2004; Marco-Jiménez et al., 2006). When observing live
spermatozoa, darkfield microscopy and stroboscope flashlight
U.S. Copyright Clearance Centre Code Statement:
is used to analyze morphological changes of the flagella and
study motility (Tsvetkova et al., 1996; Billard et al., 2000;
Cosson et al., 2000). Limited research on staining techniques
in association with light microscopy has been undertaken to
date (Howell and Butts, 1983; Wirtz and Steinmann, 2006).
The development of automated sperm morphology analysis
(ASMA) has led to an increase in number of studies on
spermatozoa of animals, mainly mammals (de Monserrat
et al., 1995; Iguer-Ouada and Verstegen, 2001; Soler et al.,
2005). Today, computerized systems enable automatic routine
analyses of sperm shapes and fosters the development of image
analysis for characterizing spermatozoa (Hidalgo et al., 2005).
The relationship between morphology and motility of
spermatozoa, and how such relations impact on fertilization,
is an intriguing scientific question when studying fish fertility
and reproduction. Sperm competition occurs when sperm
originate from two or more males, competing simultaneously
for changes to fertilize an egg. At the individual gamete level,
one would expect that sperm swimming speed will be rapid in
circumstances when the risk of sperm competition is high
(Stockley et al., 1997; Gage et al., 2002, 2004). However,
published results do not always support this assumption
(Lahnsteiner et al., 1998, Linhart et al., 2005).
The objectives of the present study, therefore, were to: (i)
develop a staining protocol for fish spermatozoa to allow
quantitative assessment of morphological characters, (ii)
demonstrate the utility of this method to characterize sperm
shape and examine inter-male variability and (iii) investigate
the possible relationship of head shape and tail length with
motility patterns. Sperm of Atlantic cod (G. morhua Linnaeus,
1758) were selected for study as an extensive literature exists on
cod reproductive biology for both natural and cultured
populations (COSEWIC 2003, Trippel, 2003) while limited
research has been conducted on cod sperm morphology
(Morrison, 1990).
Materials and methods
Atlantic cod were collected from Cape Sable, southwest Nova
Scotia and held in captivity for 5 months in a 4-m diameter
tank (volume 15 m3) at the Biological Station, St Andrews,
NB, Canada. They were maintained on a mixed diet of
Atlantic herring, squid and shrimp with the water temperature
during the winter spawning remaining at 3–4C (salinity
30 ppt). Twelve males were used in the study ranging from
49.8 to 75.9 cm fork length (FL) and 1.27 and 3.97 kg body
weight (W) (Table 1). Condition factor was calculated using
the equation, K = W ⁄ FL3 · 100 (Fulton, 1902).
0175–8659/2008/2404–0398$15.00/0
www.blackwell-synergy.com
1.88
2.24
2.48
2.74
2.76
2.67
3.23
2.75
3.51
4.90
3.97
2 56.0
3 59.3
4 60.2
5 61.0
6 62.5
7 65.5
8 66.8
9 67.5
10 67.9
11 75.7
12 75.9
S
0.91 61.5
1.14 35.0
1.12 90.0
0.89 34.5
1.08 21.5
0.95 15.5
1.13 25.0
1.21 37.0
1.03 22.5
1.07 24.0
1.52 32.0
1.03 47.0
CF
L
446 94.7 3.24
(0.24)
340 68.7 2.87
(0.18)
411 87.3 3.18
(0.24)
432 91.5 3.30
(0.23)
432 89.6 2.96
(0.18)
407 85.0 3.30
(0.20)
410 68.7 3.12
(0.23)
396 82.8 3.07
(0.23)
439 90.7 2.96
(0.19)
446 94.3 3.36
(0.19)
389 78.9 2.93
(0.23)
381 78.1 2.90
(0.18)
4929 85.5 3.11
(0.27)
n⁄%
1.95
(0.15)
1.71
(0.11)
1.78
(0.16)
1.92
(0.14)
1.77
(0.12)
1.90
(0.13)
1.78
(0.15)
1.78
(0.14)
1.72
(0.12)
1.97
(0.12)
1.74
(0.16)
1.74
(0.12)
1.82
(0.16)
W
5.42
(0.63)
4.23
(0.36)
4.91
(0.67)
5.40
(0.58)
4.46
(0.39)
5.32
(0.48)
4.77
(0.57)
4.68
(0.53)
4.39
(0.40)
5.72
(0.48)
4.38
(0.62)
4.31
(0.42)
4.86
(0.71)
A
8.83
(0.56)
7.77
(0.39)
8.56
(0.62)
8.92
(0.52)
8.00
(0.38)
8.89
(0.46)
8.37
(0.54)
8.28
(0.52)
7.98
(0.41)
9.14
(0.43)
8.09
(0.53)
7.84
(0.43)
8.42
(0.66)
P
1.67
(0.12)
1.68
(0.14)
1.79
(0.14)
1.73
(0.13)
1.68
(0.14)
1.74
(0.12)
1.76
(0.14)
1.73
(0.14)
1.72
(0.14)
1.71
(0.11)
1.69
(0.15)
1.67
(0.12)
1.72
(0.14)
Ell
Elongated head
0.87
(0.03)
0.88
(0.03)
0.84
(0.03)
0.85
(0.03)
0.87
(0.03)
0.85
(0.03)
0.86
(0.03)
0.86
(0.03)
0.87
(0.03)
0.86
(0.03)
0.86
(0.04)
0.88
(0.03)
0.86
(0.03)
Rug
0.25
(0.03)
0.25
(0.04)
0.28
(0.04)
0.27
(0.04)
0.25
(0.04)
0.27
(0.03)
0.27
(0.04)
0.26
(0.04)
0.26
(0.04)
0.26
(0.03)
0.25
(0.049
0.25
(0.03)
0.26
(0.04)
Elo
0.92
(0.04)
0.91
(0.05)
0.91
(0.05)
0.92
(0.05)
0.92
(0.04)
0.93
(0.05)
0.91
(0.05)
0.92
(0.05)
0.91
(0.04)
0.91
(0.04)
0.92
(0.05)
0.92
(0.05)
0.92
(0.05)
Reg
80.14
(4.76)
78.64
(3.81)
78.45
(2.77)
83.30
(4.58)
73.22
(2.45)
80.48
(5.24)
80.09
(3.72)
77.9
(4.15)
77.8
(2.85)
75.71
(4.59)
74.98
(4.78)
80.01
(2.62)
78.39
(4.62)
Tail
839 14.5
107 21.1
104 21.1
27 5.7
45 9.3
82 17.2
72 14.9
72 15.0
50 10.4
40 8.5
60 12.7
155 31.3
25 5.3
n⁄%
2.23
(0.18)
2.06
(0.16)
2.13
(0.18)
2.21
(0.18)
2.08
(0.17)
2.18
(0.16)
2.10
(0.18)
2.15
(0.20)
2.05
(0.15)
2.31
(0.4)
2.23
(0.17)
2.07
(0.16)
2.13
(0.18)
L
2.03
(0.16)
1.92
(0.12)
1.94
(0.14)
2.03
(0.14)
1.91
(0.11)
2.07
(0.13)
1.95
(0.15)
1.96
(0.15)
1.90
(0.11)
2.11
(0.13)
2.03
(0.16)
1.93
(0.13)
1.97
(0.15)
W
3.84
(0.46)
3.40
(0.35)
3.57
(0.39)
3.81
(0.48)
3.44
(0.32)
3.90
(0.38)
3.50
(0.44)
3.61
(0.44)
3.36
(0.32)
4.16
(0.34)
3.82
(0.52)
3.43
(0.329
3.60
(0.44)
A
7.00
(0.44)
6.57
(0.37)
6.75
(0.39)
7.00
(0.49)
6.60
(0.33)
7.06
(0.35)
6.68
(0.45)
6.81
(0.45)
6.55
(0.37)
7.30
(0.30)
7.08
(0.48)
6.59
(0.33)
6.78
(0.45)
P
1.10
(0.07)
1.08
(0.09)
1.10
(0.10)
1.10
(0.08)
1.09
(0.09)
1.05
(0.08)
1.08
(0.09)
1.10
(0.10)
1.08
(0.08)
1.10
(0.07)
1.10
(0.08)
1.08
(0.09)
1.09
(0.09)
Ell
Round head
0.98
(0.02)
0.98
(0.03)
0.98
(0.02)
0.98
(0.03)
0.99
(0.02)
0.98
(0.02)
0.98
(0.03)
0.98
(0.02)
0.98
(0.03)
0.98
(0.02)
0.95
(0.04)
0.99
(0.02)
0.98
(0.03)
Rug
0.05
(0.02)
0.04
(0.03)
0.05
(0.04)
0.04
(0.03)
0.05
(0.03)
0.04
(0.03)
0.04
(0.03)
0.05
(0.04)
0.04
(0.03)
0.05
(0.03)
0.05
(0.03)
0.04
(0.03)
0.05
(0.03)
Elo
0.93
(0.05)
0.92
(0.05)
0.91
(0.05)
0.93
(0.05)
0.91
(0.06)
0.91
(0.04)
0.92
(0.05)
0.92
(0.05)
0.91
(0.04)
0.92
(0.04)
0.93
(0.05)
0.91
(0.05)
0.92
(0.05)
Reg
n
80.15
49
(2.72)
76.76
38
(3.01)
80.62
40
(2.93)
82.50
27
(2.80)
74.07
35
(2.64)
81.22
20
(2.99)
80.86
50
(2.39)
77.36
36
(2.91)
78.18
32
(4.25)
80.47
10
(4.61)
76.70
29
(4.20)
80.25
10
(3.69)
79.10 376
(3.96)
Tail
n
71.7
52
(30.5)
72.3
82
(38.4)
76.4
50
(39.9)
80.9
11
(37.7)
78.5
27
(29.1)
95.2
25
(40.3)
86.4
65
(34.6)
79.2
31
(34.5)
74.9
21
(28.6)
54.8
1
(33.3)
83.2
41
(40.3)
75.6
8
(29.9)
76.8
414
(35.4)
30 s
Sperm motility
60.4
(30.5)
65.5
(25.5)
64.9
(33.1)
48.3
(14.2)
72.9
(30.3)
38.9
(7.3)
60.8
(29.8)
64.8
(21.9)
79.9
(37.2)
64.1
())
65.7
(23.8)
73.2
(33.4)
63.3
(10.9)
70 s
A, area in lm2; CF, condition factor; Ell, ellipticity; Elo, elogation; FL, furcal length in cm; L, length in lm; P, perimeter in lm; Reg, regularity; Rug, rugosity; S, spermatocrit in %; W, body weight in kg; W, width in lm; %, percentage
of head type.
Total 64.2 2.87
(7.5) (0.96)
1.27
W
1 49.8
Code FL
Fish
Sperm morphology
Table 1
Characteristics of the fishes used in the study, shape parameters and length tail (means and standard deviation) of the head types noted (elongated and round) for each individual and total
Sperm morphology in Atlantic cod
399
400
Semen was sampled by stripping ripe males from 27
February to 10 March 2006. Care was taken not to use sperm
samples that were contaminated by either feces or urine. Fish
was anaesthetized using tricaine methanesulphonate (MS 222
at 1 : 20 000 dilution), the external genital duct region was
wiped dry and semen was expressed by applying slight pressure
along the abdomen. The first portion of the male ejaculate was
watery and was discarded. Semen was collected in 100-mL
glass beakers covered with wax paper and held in a refrigerator
at 4C (Trippel, 2003).
A small volume of semen (10 lL) was diluted at a 1 : 100
ratio with a medium composed of seawater and 3% citrate
sodium (1 : 1). The diluted semen was deposited in an
Eppendorf tube and gently shaken for 30 s. For each individual, five semen smears per ejaculate were each prepared as
follows: 5 lL aliquot of semen was placed on a slide and
spread out into a smear with the edge of a second slide and air
dried for 10 s. The smears were stained with Hemacolor (EMD
Chemicals, Inc., Gibbstown, NJ) such that each slide was
immersed in fixative for 5 s, rinsed in colorant A for 3 min and
in colorant B for 1 s (this was repeated four times). Each slide
was then washed in distilled water to eliminate excess stain and
allowed to air dry. Once dried, all the slides were permanently
sealed with Eukitt mounting medium (Kindler & Co., Freiburg, Germany) and topped with a coverslip. Morphometric
analyses of the spermatozoa heads were performed using the
morphological module of the sperm image analysis software
ISAS (Proiser R+D SL, Buñol, Spain). The equipment
consisted of a microscope (Leica DMLB, Tokyo, Japan) with a
100· objective, a 0.67· photo-ocular and a digital video
camera (Balser A310, Basler AG, Germany). A total of 100
spermatozoa heads of each smear were digitized at random in
different fields with a 100· oil immersion objective. This
process was performed by manual selection to avoid the
inclusion of foreign particles that may have interfered with the
posterior image processing. The digitized cells were automatically segmented with a range of gray-level values predetermined by the analysis factor (the automatic algorithm used to
define the contrast between cell and background). The system
detected the boundary of sperm heads being displayed as white
overlays superimposed on the microscopic video image. Those
cells that could not have their boundary clearly delimited were
excluded. The size parameters estimated automatically by
ISAS were: length (L, in lm), width (W, in lm), area (A, in
lm2) and perimeter (P, in lm), the shape indices: elipticity
(L ⁄ W), rugosity (4pA ⁄ P2), elongation [(L – W) ⁄ (L + W)]
and regularity (pLW ⁄ 4A).
Flagellum length was measured from its insertion in the
head to the end of the filament. Ten spermatozoa of each head
type (elongated and round) per male were measured at 40·
magnification (Burness et al., 2004). The Imagen-Pro Plus
(Media Cybernetics, Inc.) software was used for sperm tail
measurements.
Spermatocrit (percent of packed cellular material in semen
after centrifugation) was used to estimate spermatozoa density
as it is a reliable indicator of sperm density in cod (Rakitin
et al., 1999). It was measured in triplicate for each semen
sample by centrifuging undiluted milt samples for 15 min at
7500 rpm in a microhematocrit centrifuge and by reading
sperm cell density against a calibrated scale.
To analyse motility, semen dilutions (1 : 3000 semen–
seawater) were made by placing a 10-lL aliquot of semen in
a 30-mL glass beaker containing seawater (4C at salinity
30 ppt). Semen and seawater were then gently shaken for 5 s.
V. M. Tuset, E. A. Trippel and J. de Monserrat
Sperm movement was analyzed with the same equipment as
described for morphology using the motility module of ISAS
and a light microscope (magnification 40·, Leica DMLB,
Japan). The images were captured 20 s after sperm were
mixed. The experiments were not made in a cold room.
Although cod sperm can remain motile for up to 60 min
(Trippel and Morgan, 1994), the motility study concentrated
on sperm movements in the first minutes post-activation with
sea water because this is likely the most important period to
achieve fertilization (Hutchings et al., 1999; Rakitin et al.,
2001; Bekkevold et al., 2002). Therefore, the 20 s delay in the
start of sperm observations was unlikely to affect our
comparative estimates of sperm motility (Litvak and Trippel,
1998). For each male, curvilinear velocity (VCL, in lm s)1),
linearity (LIN, curvilinear velocity ⁄ straight line velocity · 100), straightness, (straight line velocity ⁄ average velocity · 100) mean amplitude of lateral head displacement (ALH,
in lm), wobble of the curvilinear trajectory (WOB,
VAP ⁄ VCL · 100) and beat cross frequency (BCF, Hz) of
swimming paths over ten 5 s intervals in different fields during
50 s were recorded. The beginning of the movement was
defined at 30 s from its activation and the end at 70 s. Sperm
was considered to be motile if their progressive motility had
straight line velocity over 4 lm s)1.
To identify head morphological variations, a multivariate
analysis involving four steps was used (Martinez-Pastor et al.,
2005; Nuñez-Martı́nez et al., 2006). Firstly, the aim was to
study the head shape as a whole and to discern the particular
contribution of each variable to the total variance shape. This
was done by principal component analysis (PCA) where the
component matrix was evaluated and extracted and the
orthogonal factors were rotated used the Varimax criteria.
Consequently, new factors were created as combinations of
original variables. The second step was to perform a nonhierarchical analysis using the k-means model that used
Euclidean distances to estimate the cluster centres selecting
the variables based on the PCA. The procedure selected 13
clusters with some outliers detected which were removed and
the procedure rerun. The third step was to apply hierarchical
clustering using the average linkage method (UPGMA).
Finally, a stepwise discriminant analysis of the clusters was
obtained to reduce the number of clusters and to aid in their
interpretation (SPSS; SPSS Inc.).
For each morphometric variable (head or tail), normality of
the data distributions and variance homogeneity were checked
by Kolmogorov–Smirnov and Levene tests, respectively. If the
data fulfilled these premises, comparison of samples was
performed with StudentÕs t-test (two samples) or one-way
ANOVA (more than two samples) using a post hoc TukeyÕs test;
if the assumptions were not met a Kruskal–Wallis test was
carried out. Linear relationships among fish body characteristics, sperm size and motility were calculated. Data in
percentage (spermatocrit, linearity, straightness and wobble)
were arcsine transformed (Zar, 1996).
Results
Of the 6000 sperms studied, 96.1% were correctly analyzed for
head shape using ISAS. The first two principal components
of the PCA performed on these data explained 85.3% of the
variation in head shape. PRIN1 explained 59.8% of the
variance and represented length, elongation, rugosity, ellipticity and perimeter, while PRIN2 explained 25.4% and mainly
represented width. PRIN1 represented sperm having an
Sperm morphology in Atlantic cod
elongated head and PRIN2 represented those with a round
head. The 13 initial clusters were grouped into two revealing the
presence of elongated and round heads (Fig. 1). The discriminant functions were formed with elongation, ellipticity, perimeter, rugosity and area correctly assigning 99.5% of cases.
Average percentage of sperm with an elongated head varied
between 68.7 and 94.7% among males. Ninety-five percent of
elongated heads measured between 2.6 and 3.6 lm in length,
and 1.5 and 2.1 lm in width; while the round heads ranged
between 1.8 and 2.5 lm in length, and 1.7 and 2.3 lm in width
(Table 1). Within males, mean head length of elongated sperm
was greater than round, and the opposite trend existed for
head width. The head types showed significant differences in all
shape characteristics except regularity (t-test: P = 0.270;
Fig. 2). Variability among males was detected for all head
attributes (K–W test: P < 0.001).
Flagellum length varied between 67.2 and 90.5 lm among
males and although males differed significantly in tail length
(ANOVA: F11,239 = 8.999, P < 0.001) this was not in relation
to head type (round ⁄ elongated) (t = 1.397; d.f. = 258,
P > 0.05). Three groups of males were identified in relation
to tail length: (i) short, individual 5; (ii) long, individual 4 and
(iii) rmedium, the latter being most common. A significant
positive relationship existed between head length and tail
length (r2 = 0.423, P < 0.05) (Fig. 3). Mean tail length was,
approximately, 96% of the total sperm length.
Mean swimming speeds varied between 54.8–95.2 lm s)1
after of sperm activation (30 s) and ranged from 48.3–
401
79.9 lm s)1 from at the end of movement (70 s) (Table 2),
achieving maximum values of 151–201 lm s)1. Male body
length and weight were not correlated with sperm morphology
and swimming speed, however, condition factor was negatively
correlated with straightness of path (r2 = 0.340 at 30 s,
r2 = 0.648 at 70 s) and wobble of the curvilinear trajectory
(r2 = 0.404 at 70 s). At the beginning of the movement (30 s),
swimming speed was negatively correlated with spermatocrit
(r2 = 0.650) and positively with wobble (r2 = 0.607) and
linearity (r2 = 0.523). At the end of tracking (70 s) the
amplitude of lateral head had increased (r2 = 0.497) and
individual males with short sperm maintained their velocity for
a longer period (r2 = 0.380). Sperm movement began in a
linear direction and over time this was lessened such that the
amplitude inversely related with linearity, straightness and
wobble (Table 3, Fig. 4).
(a)
Fig. 2. Comparison of shape parameter between head types. Data
represents mean ± standard deviation. Different letters indicate significant differences (StudentÕs t-test: P < 0.05)
(b)
Fig. 1. Head types of Atlantic cod sperm. (a) Hemacolor 40·; (b)
phase contrast 100· oil immersion
Fig. 3. Relationship between mean of head and tail length (lm). The
line was fitted by y = 55.918 + 7.685x, r2 = 0.423, P < 0.05
n
49
38
40
27
35
20
50
36
32
10
29
10
Code
1
2
3
4
5
6
7
8
9
10
11
12
71.7
72.3
76.4
80.9
78.5
95.2
86.4
79.2
74.9
54.8
83.2
75.6
VCL
(30.5)
(38.4)
(39.9)
(37.7)
(29.1)
(40.3)
(34.6)
(34.5)
(28.6)
(33.3)
(40.3)
(29.9)
78.7
62.2
69.4
68.3
74.0
79.2
85.3
79.5
83.1
54.6
82.0
69.2
LIN
(15.6)
(25.6)
(20.9)
(23.9)
(20.8)
(16.8)
(10.1)
(19.8)
(13.8)
(25.8)
(17.4)
(19.9)
90.8
74.5
84.3
79.5
86.6
87.7
93.1
87.4
92.5
73.7
90.5
84.5
STR
(11.5)
(22.1)
(17.7)
(20.7)
(16.7)
(13.2)
(6.3)
(17.5)
(6.3)
(22.9)
(9.6)
(16.5)
1.9
1.9
2.01
2.1
2.1
2.0
1.8
1.7
1.6
1.9
1.8
1.8
(0.5)
(0.5)
(0.9)
(0.7)
(0.6)
(0.5)
(0.7)
(0.5)
(0.6)
(0.6)
(0.5)
(0.4)
ALH
86.0
80.8
81.2
84.2
83.8
89.3
91.6
89.9
89.3
71.7
89.6
80.4
(10.4)
(16.9)
(13.4)
(12.9)
(12.4)
(9.2)
(6.3)
(10.7)
(10.5)
(15.8)
(13.0)
(11.3)
WOB
8.4
8.1
8.8
7.5
7.3
8.1
9.1
7.9
7.8
7.5
7.3
7.3
(2.9)
(2.6)
(2.9)
(2.3)
(2.9)
(3.3)
(2.4)
(2.8)
(2.3)
(2.5)
(2.9)
(2.8)
BCF
52
82
50
11
27
25
65
31
21
1
41
8
n
70 s
60.4
65.5
64.9
48.3
72.9
38.9
60.8
64.8
79.9
64.1
65.7
73.2
VCL
(30.5)
(25.5)
(33.1)
(14.2)
(30.3)
(7.3)
(29.8)
(21.9)
(37.2)
())
(23.8)
(33.4)
83.2
63.0
83.4
83.8
79.7
82.0
83.2
84.2
94.8
90.2
84.8
81.6
LIN
(13.0)
(29.8)
(11.3)
(15.9)
(16.3)
(16.6)
(11.7)
(8.7)
(3.9)
())
(14.0)
(14.3)
93.4
81.9
93.5
92.4
90.7
92.5
94.7
92.2
94.8
96.2
94.5
94.8
STR
(6.1)
(19.1)
(4.1)
(8.1)
(8.2)
(10.9)
(3.9)
(5.8)
(3.9)
())
(5.1)
(4.1)
1.4
2.0
1.4
1.3
1.7
1.0
1.3
1.4
1.5
1.3
1.3
1.6
(0.5)
(0.9)
(0.5)
(0.5)
(0.5)
(0.2)
(0.4)
(0.4)
(0.3)
())
(0.3)
(0.5)
ALH
88.5
72.3
89.1
89.8
87.2
88.2
87.7
91.3
89.9
93.8
89.2
85.7
(11.5)
(24.3)
(10.4)
(11.4)
(12.9)
(12.9)
(11.1)
(7.4)
(6.8)
())
(12.6)
(12.1)
WOB
9.8 (2.6)
8.3 (3.6)
10.1 (3.3)
7.4 (2.5)
8.4 (3.1)
9.9 (2.8)
10.1 (3.1)
10.2 (3.1)
8.2 (2.9)
10.0 ())
10.2 (2.7)
10.1(1.3)
BCF
30
70
30
70
30
70
30
70
30
70
30
70
s
s
s
s
s
s
s
s
s
s
s
s
0.115
0.278
)0.381
0.705
0.325
0.252
)0.413
)0.331
0.110
)0.543
0.023
)0.040
)0.512
)0.533
0.220
)0.222
0.006
)0.155
30 s
BCF
0.201
0.139
0.064
)0.063
)0.248
)0.366
70 s
)0.022
)0.157
0.723
)0.086
)0.381
)0.348
0.334
0.316
30 s
LIN
0.148
)0.148
)0.192
)0.024
)0.045
)0.670
)0.151
0.360
70 s
)0.035
)0.248
0.536
0.139
)0.466
)0.287
0.308
0.305
0.936
0.265
30 s
STR
0.279
)0.071
)0.126
0.085
)0.165
)0.593
)0.076
0.478
0.220
0.933
70 s
0.008
)0.106
0.779
)0.159
)0.398
)0.312
0.305
0.133
0.952
0.020
0.840
0.061
30 s
WOB
0.044
)0.220
)0.166
)0.084
0.023
)0.682
)0.155
0.298
0.134
0.976
0.230
0.841
70 s
)0.232
)0.427
0.174
)0.616
0.267
)0.500
0.381
0.035
)0.030
0.174
)0.062
0.221
0.016
0.137
Sperm size
0.220
0.226
)0.806
0.248
)0.052
0.066
)0.443
0.173
)0.641
0.375
)0.470
0.377
)0.757
0.299
)0.104
S
)0.151
0.303
)0.101
)0.123
0.228
0.439
)0.060
)0.254
)0.398
)0.718
)0.573
)0.836
)0.291
)0.641
)0.417
)0.032
CF
)0.454
)0.535
)0.430
)0.051
0.283
)0.334
)0.273
)0.248
)0.264
0.578
)0.089
0.385
)0.320
0.642
0.100
0.412
)0.282
Elongated%
ALH, amplitude of lateral head displacement; BCF, beat cross frequency; CF, condition factor; FL, furcal length; LIN, linearity; S, spermatocrit; STR, straightness; VCL, curvilinear velocity; W, body
weight; WOB, wobble of the curvilinear trajectory; 30 s beginning at movement; 70 s end of movement; bold values are the significant regressions.
Sperm size
S
CF
WOB
STR
LIN
BCF
ALH
FL
W
VCL
70 s
30 s
30 s
70 s
ALH
VCL
Table 3
Correlation matrix among fish parameters, sperm size (head plus tail) and motility
ALH, lateral head displacement (lm); BCF, beat cross frequency (Hz); LIN, linearity (%); STR, straightness (%); VCL, curvilinear speed (lm s)1); WOB, wobble of the curvilinear trajectory (%).
30 s
Fish
Table 2
Characteristics of the parameters of motility (means and standard deviation) at the beginning (30 s after activation) and the end (70 s after activation) for each individual
402
V. M. Tuset, E. A. Trippel and J. de Monserrat
Sperm morphology in Atlantic cod
403
Fig. 4. Relationships of swimming speed with spermatocrit (arcsine), sperm size and condition factor at the beginning and at the end of movement
Discussion
This study has demonstrated that the head shape of fish sperm
can be analyzed using the same procedures as applied in others
animals in relation to staining techniques and automated
morphology analysis. Hemacolor proved to be a good staining
technique and accurately defined the boundary of the head
permitting quantitative assessment using the ISAS system. In
addition, the definition of the tail boundary presented an
excellent contrast with the background sharp. To achieve
straight tails enabling easier assessment of length (mean
79.1 ± 4.5 lm) were used seawater to active sperm and this
resulted in good spatial distribution, but the seawater also
increased the number of cells swelling. The development of this
technique was not simple and we incorporated many tests with
different extenders and times to dry, fix and colour. Some
artefacts appeared, although a broad area of the slide was
always available for suitable analysis. Previous studies (Howell
and Butts, 1983; Bastardo et al., 2004; Wirtz and Steinmann,
2006) have also described problems with staining protocol and
the general lack of in-depth studies of sperm head morphology
suggest technique development has been an issue.
Two head pleimorphims of sperm of Atlantic cod were
reported with the elongated type predominating. Although
staining methods may produce morphological alterations or
mask nuclear defects, the abnormalities in size and shape are
better detected with these techniques (Chacón, 2001). The
abnormality observed, round head, is a common type in other
animals, leopard (Jayaprakash et al., 2001), alpaca (Buendia
et al., 2002) and catlle (Chenoweth, 2005), and therefore was
not an artefact produced by our techniques.
The studies performed on the fine structure of sperm of
Merluccius merluccius (Merlucciidae) (Medina et al., 2003),
Merluccius polli (Merlucciidae) (Mattei, 1991), Lota lota
(Gadidae) (Lahnsteiner et al., 1994) and G. morhua (Gadidae)
(Morrison, 1990) reveal that head shape in these species is
elongated. Morphologically, the spermatozoa of Gadiformes
have been classified as type II, asymmetrical insertion of
flagellum, nulear fossa and with the centrioles close to the
nucleus (Mattei, 1991; Lahnsteiner et al., 1994; Medina et al.,
2003). However, the spermatozoa of G. morhua have asymmetry of the flagellum, nuclear fossa and the centrioles are
inside it (Morrison, 1990). In addition, the flagellum length is
very larger (79 lm; present study) compared with the size of
M. merluccius (30 lm; Medina et al., 2003). Consequently, the
spermatozoa of Atlantic cod show a shape intermediate
between types I and II, which has interesting phylogenetic
implications.
Size and shape of spermatozoa are known to be associated
with the fertilization process (Sailer et al., 1996; Hirai et al.,
2001). These differences between males may be affect the
outcome of sperm competition. Some studies showed that long
404
sperm swim faster and than short sperm and a trade-off exists
between mean sperm mean and ejaculate longevity (Stockley
et al., 1997; Levitan, 2000; Jobling et al., 2003; Burness et al.,
2004); while others found no evidence of these (Gage et al.,
1998, 2002; Vladić et al., 2002; Casselman et al., 2006). Our
study showed a trade-off between sperm size and speed and
noted that spermatocrit as a factor more important than size
sperm in its correlation with initial speed. Rakitin et al. (1999),
in Atlantic cod, concluded that spermatocrit and male condition factor affect were related to fertilization success of males;
while sperm motility did not explain the differences in
fertilization success among males, although they suggested
that differences in swimming velocity might only be important
if two spermatozoa arrive at simultaneously the micropyle. In
Atlantic salmon, Vladić et al. (2002) demonstrated that the
longer spermatozoa have more ATP available and greater
fertilization success; and Gage et al. (2004) noted males with
faster swimming sperm fertilized a greater proportion of eggs.
Furthermore, this suggests that the longest spermatozoa swim
fastest and their energy and not longevity. Casselman et al.
(2006) found no correlation between sperm morphology and
sperm swimming speed in walleye but suggested the relationship exists in bluegill sunfish. Consequently, the various studies
are not in agreement, however, we concur with these authors
when they assert that swimming speed is influenced by more
factors than sperm length (as we have demonstrated in this
study), although we believe sperm length could be important.
Most of these studies have been performed using in freshwater
fish which typically have a relatively brief activity period
compared to marine species. For this reason, we recommend
further studies of this nature be conducted on marine fishes.
Spermatozoa of cod in the Baltic Sea have been reported to
be motile for 16 min on average (Westin and Nissling, 1991)
and for even longer periods for those of Atlantic cod such that
they retained about 50% of their initial fertilization capacity
after 60 min in sea water (Trippel and Morgan, 1994). Trippel
and Neilson (1992) did not find a relationship between egg
fertilization rate and sperm motility measured in seawater,
however, Litvak and Trippel (1998) demonstrated that motility
is influenced by ovarian fluid and suggested that vibrating and
(or) motionless sperm became activated when in the presence
of an egg. Comparing changes in swimming speed over time of
individuals with short and long sperm, revealed very different
patterns (Fig. 5). Long sperm achieve their maximum speed at
Fig. 5. Lineal comparison of motility pattern of spermatozoa between
one individual of short sperm (solid line and dark circles) and long
sperm (hatched line and white squared)
V. M. Tuset, E. A. Trippel and J. de Monserrat
the beginning and rapidly decrease whereas short reach similar
initial speeds and do not decline as rapidly. Insufficient data
exist to make widespread conclusions. Considering the results
of this study, further research is required including many
factors (e.g. sperm density, morphology, motility activate by
ovarian fluid, and successful fertilization) to help to discern the
importance of sperm morphology in the fertilization process.
Acknowledgements
We thank Marc for excellent technical assistance. The authors
gratefully acknowledge suggestions and criticisms of anonymous reviewers that greatly improved the manuscript. This
project was funded by grant from Government of the Canary
Islands.
References
Asturiano, J. F.; Marco-Jiménez, F.; Pérez, L.; Balasch, S.; Garzón,
D. L.; Peñaranda, D. S.; Vicente, J. S.; Viudes de Castro, M. P.;
Jover, M., 2006: Effects of hCG as spermiation inducer on
European eel semen quality. Theriogenology 66, 1012–1020.
Balshine, S.; Leach, B. J.; Neat, F.; Werner, N. Y.; Montgomerie, R.,
2001: Sperm size of African cichlids in relation to sperm
competition. Behav. Ecol. 12, 726–731.
Bastardo, H.; Guedez, C.; León, M., 2004: Caracterı́sticas del semen de
trucha arco iris de diferentes edades, bajo condiciones de cultivo
en Mérida, Venezuela. Zoot. Trop. 22, 277–288.
Bekkevold, D.; Hansen, M. M.; Loeschcke, V., 2002: Male reproductive competition in spawning aggregations of cod (Gadus morhua,
L.). Mol. Ecol. 11, 91–102.
Billard, R.; Cosson, J.; Linhart, O., 2000: Changes in the flagellum
morphology of intact and frozen ⁄ thawed siberian sturgeon
Acipenser baeri Brandt sperm motility. Aquacult. Res. 30, 1–5.
Buendia, P.; Soler, C.; Paolocchi, F.; Gago, G.; Urquieta, B.; PérezSánches, F.; Bustos-Obregrón, E., 2002: Morphometric characterization and classification of alpaca sperms heads using the
sperm-class analyzer computer-assisted system. Theriogenology
57, 1207–1218.
Burness, G.; Schulte-Hostedde, A. I.; Casselman, S. J.; Moyes, C. D.;
Montgomerie, R., 2004: Sperm swimming speed and energetics
vary with sperm competition risk in bluegill (Lepomis macrochirus). Behav. Ecol. Sociobiol. 56, 65–70.
Casselman, S. J.; Schulte-Hostedde, A. I.; Montgomerie, R., 2006:
Sperm quality influences male fertilization success in walleye
(Sander vitreus). Can. J. Fish. Aquat. Sci. 63, 2119–2125.
Chacón, J., 2001: Assessment of sperm morphology in zebu bulls,
under field conditions in the tropics. Rep. Dom. Anim. 36, 91.
Chenoweth, P., 2005: Genetic sperm defects. Theriogenology 64, 457–
468.
COSEWIC, 2003: COSEWIC assessment and update status report on
the Atlantic cod Gadus morhua in Canada. Committee on the
Status of Endangered Wildlife in Canada, Ottawa, p. 76.
Cosson, J.; Linhart, O.; Mims, S.; Shelton, W.; Rodina, M., 2000:
Analysis of motility parameters from paddlefish (Polyodon
spathula) and shovelnose sturgeon (Scaphirhynchus platorynchus)
spermatozoa. J. Fish Biol. 56, 1348–1367.
Fulton, T., 1902: Rate of growth of seas fishes. Sci. Invest. Fish. Div.
Scot. Rept. 20, 1035–1039.
Gage, M. J. G.; Stockley, P.; Parker, G. A., 1998: Sperm morphometry
in the Atlantic salmon. J. Fish Biol. 53, 835–840.
Gage, M. J. G.; MacFarlane, C.; Yeates, S.; Shackelton, R.; Parker,
G. A., 2002: Relationship between sperm morphometry and
sperm motility in the Atlantic salmon. J. Fish Biol. 61, 1528–1539.
Gage, M. J. G.; Macfarlane, C. P.; Yeates, S.; Ward, R. G.; Searle,
J. B.; Parker, G. A., 2004: Spermatozoal traits and sperm
competition in Atlantic salmon: relative sperm velocity is
the primary determinant of fertilization success. Curr. Biol. 14,
44–47.
Hidalgo, M.; Rodrı́guez, I.; Dorado, J.; Sanz, J.; Soler, C., 2005: Effect
of sample size and staining methods on stallion sperm morphometry by the Sperm Class analyzer. Vet. Med. 50, 24–32.
Hirai, M.; Boersma, A.; Hoeflich, A.; Wolf, E.; Föll, J.; Aumüller, R.;
Braum, J., 2001: Objectively measured sperm motility and sperm
Sperm morphology in Atlantic cod
head morphometry in boars (Sus scrofa): relation to fertility and
seminal plasma growth factors. J. Androl. 22, 104–110.
Howell, W. M.; Butts, D., 1983: Silver staining of spermatozoa from
living and preserved museum fishes: a new taxonomic approach.
Copeia 4, 974–978.
Hutchings, J. A.; Bishop, T. D.; McGregor-Shaw, C. R., 1999:
Spawning behaviour of Atlantic cod, Gadus morhua: evidence of
mate competition and mate choice in a broadcast spawner. Can.
J. Fish. Aquat. Sci. 56, 97–104.
Iguer-Ouada, M.; Verstegen, J. P., 2001: Validation of the sperm
quality analyzer (SQA) for dog sperm analysis. Theriogenology
55, 1143–1158.
Jamieson, B. G. M. 1991: Fish evolution and systematics: evidence
from spermatozoa. University Press, Cambridge, p. 319.
Jayaprakash, D.; Patil, S. B.; Kumar, M. N.; Majumdar, K. C.;
Shivaji, S., 2001: Semen characteristics of the captive Indian
leopard, Panthera pardus. J. Androl. 22, 25–33.
Jobling, S.; Coey, S.; Whitmore, J. G.; Kime, D. E.; Van Look, K. J.
W.; McAllister, B. G.; Beresford, N.; Henshaw, A. C.; Brighty,
G.; Tyler, C. R.; Sumpter, J. P., 2003: Wild intersex roach
(Rutilus rutilus) have reduced fertility. Biol. Reprod. 67, 515–
524.
Kime, D. E.; Van Look, K. J.; McAllister, B. G.; Huyskens, G.;
Rurangwa, E.; Ollevier, F., 2001: Computer-assisted sperm
analysis (CASA) as tool for monitoring sperm quality in fish.
Comp. Biochem. Physiol. 130, 425–433.
Lahnsteiner, F.; Mansour, N., 2004: Sperm fine structure of the
pikeperch, Sander lucioperca and its differences from Perca
fluviatilis (Percidae). J. Submicr. Pathol. Cytol. 36, 309–312.
Lahnsteiner, F.; Weismann, T.; Patzner, R. A., 1994: Fine structure of
spermatozoa of the turbot, Lota lota (Gadidae, Pisces).
J. Submicr. Cytol. Pathol. 26, 445–447.
Lahnsteiner, F.; Berger, B.; Weismann, T.; Patzner, R. A., 1998:
Determination of semen quality of the rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and
spermatozoal metabolism. Aquaculture 163, 163–181.
Levitan, D. R., 2000: Sperm velocity and longevity trade off each other
and influence fertilization in the sea urchin Lytechinus variegates.
Proc. R. Soc. Lond., B, Biol. Sci. 267, 531–534.
Linhart, O.; Rodina, M.; Gela, G.; Kocour, M.; Vandeputte, M., 2005:
Spermatozoal competition in common carp (Cyprinus carpio):
what is the primary determinant of competition success? Reproduction 130, 705–711.
Litvak, M. K.; Trippel, E. A., 1998: Sperm motility patterns of
Atlantic cod (Gadus morhua) in relation to salinity: effects of
ovarian fluid and egg presence. Can. J. Fish. Aquat. Sci. 55, 1871–
1877.
Marco-Jiménez, F.; Pérez, L.; Viudes de Castro, M. P.; Garzón, D. L.;
Peñaranda, D. S.; Vicente, J. S.; Jover, M.; Asturiano, J. F., 2006:
Morphometry characterisation of European eel spermatozoa with
computer-assisted spermatozoa analysis and scanning electron
microscopy. Theriogenology 65, 1302–1310.
Martinez-Pastor, F.; Garcı́a-Macias, V.; Alvarez, M.; Herráez, P.;
Anel, L.; de Paz, P., 2005: Sperm subpopulation in Iberian red
deer epidydimal sperm and their changes through the cryopreservation process. Biol. Reprod. 72, 316–327.
Mattei, X., 1991: Spermatozoon ultrastructure and its systematic
implications in fishes. Can. J. Zool. 69, 3038–3055.
Medina, A.; Megina, C.; Abascal, F. J.; Calzada, A., 2003: The sperm
ultrastructure of Merluccius merluccius (Teleostei, Gadiformes):
phylogenetic considerations. Acta Zool. 84, 131–137.
de Monserrat, J. J.; Perez-Sanchez, F.; Tablado, L.; Soler, C., 1995:
The Sperm-Class Analyzer: a new automated system for human
405
sperm morphometry and classification. Contracept. Fert. Sex. 23,
S126.
Morrison, C. M., 1990: Histology of Atlantic cod, Gadus morhua: an
Atlas. Part 3. Reproductive tract. Can. Spec. Publ. Fish. Aquat.
Sci. 100, 1–177.
Nuñez-Martı́nez, I.; Moran, J. M.; Peña, F. J., 2006: A three-step
statistical procedure to identify sperm kinematic subpopulations
in canine ejaculates: changes after cryopreservation. Reprod.
Dom. Anim. 41, 408–415.
Rakitin, A.; Ferguson, M. M.; Trippel, E. A., 1999: Spermatocrit and
spermatozoa density in Atlantic cod (Gadus morhua): correlation
and variation during the spawning season. Aquaculture 170, 349–
358.
Rakitin, A.; Ferguson, M. M.; Trippel, E. A., 2001: Male reproductive
success and body size in Atlantic cod Gadus morhua L. Mar. Biol.
138, 1077–1085.
Rurangwa, E.; Kime, D. E.; Ollevier, F.; Nash, J. P., 2004: The
measurements of sperm motility and factors affecting sperm
quality in cultured fish. Aquaculture 234, 1–28.
Sailer, B. L.; Jost, L. K.; Evenson, D. P., 1996: Bull sperm head
morphometry related to abnormal chromatin structure and
fertility. Cytometry 24, 167–173.
Soler, C.; Gadea, B.; Soler, A. J.; Fernandez-Santos, M. R.; Esteso, M.
C.; Nuñez, J.; Moreira, P. N.; Nuñez, M.; Gutierrez, R.; Sancho,
M.; Garde, J. J., 2005: Comparison of three different staining
methods for the assessment of epididymal red deer sperm
morphometry by computerized analysis with ISAS. Theriogenology 64, 1236–1243.
Stockley, P.; Gage, M. J. G.; Parker, G. A.; Møller, A. P., 1997: Sperm
competition in fish: the evolution of testis size and ejaculate
characteristics. Am. Nat. 149, 933–954.
Trippel, E. A., 2003: Estimation of male reproductive success of
marine fishes. J. Northw. Atl. Fish. Sci. 33, 81–113.
Trippel, E. A.; Morgan, M. J., 1994: Sperm longevity in Atlantic cod
(Gadus morhua). Copeia 1994, 1025–1029.
Trippel, E. A.; Neilson, J. D., 1992: Fertility and sperm quality of
virgin and repeat-spawning Atlantic cod (Gadus morhua) and
associated hatching success. Can. J. Fish. Aquat. Sci. 49, 2118–
2127.
Tsvetkova, L. I.; Cosson, J.; Linhart, O.; Billard, R., 1996: Motility &
fertilizing capacity of fresh & frozen-thawed spermatozoa in
sturgeons (Acipenser baeri & A. Ruthenus). J. Appl. Ichtyol. 12,
107–112.
Van-Look, K. J. W.; Kime, D. E., 2003: Automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm.
J. Fish Biol. 63, 1020–1033.
Vladić, T. V.; Afzelius, B. A.; Bronnikov, G. E., 2002: Sperm quality as
reflected through morphology in salmon alternative life histories.
Biol. Reprod. 66, 98–105.
Westin, L.; Nissling, A., 1991: Effects of salinity on spermatozoa
motility, percentage of fertilized eggs and egg development of
Baltic cod (Gadus morhua), and implications for cod stock
fluctuations in the Baltic. Mar. Biol. 108, 5–9.
Wirtz, S.; Steinmann, P., 2006: Sperm characteristics in perch Perca
fluviatilis L. J. Fish Biol. 68, 1896–1902.
Zar, J. H. 1996. Biostatistical analysis. Prentice-Hall International,
Upper Saddle River, NJ, p. 662.
AuthorÕs address: Vı́ctor M. Tuset, Departamento de Biologı́a
Pesquera, Instituto Canario de Ciencias Marinas,
P.O. Box. 56, E-35200 Telde (Las Palmas), Canary
Islands, Spain.
E-mail: [email protected]