219
Sperm morphology, ATP content, and analysis of
motility in Atlantic halibut (Hippoglossus
hippoglossus)
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Sayyed Mohammad Hadi Alavi, Ian A.E. Butts, Azadeh Hatef, Maren Mommens,
Edward A. Trippel, Matthew K. Litvak, and Igor Babiak
Abstract: Spermatozoon of Atlantic halibut (Hippoglossus hippoglossus (L., 1758)) is uniflagellated, lacks an acrosome,
and is differentiated into a head, midpiece, and flagellum. There are two to five mitochondria in the midpiece, as well as
proximal and distal centrioles. The flagellum consisted of 9 + 2 microtubules surrounded by plasma membrane, which is
extended at the proximal part of the flagellum owing to the presence of vacuoles. After sperm activation in seawater,
sperm motility and velocity decreased from 98.4% ± 3.4% and 170.3 ± 8.9 mms–1 at 15 s after sperm activation to 4.8% ±
4.7% and 9.2 ± 8.9 mms–1 at 120 s after sperm activation, respectively. ATP content (nmolL–1 ATP per 108 spermatozoa)
significantly decreased at 60 s after sperm activation (5.9 ± 1.5) compared with at 0 and 30 s after sperm activation
(14.9 ± 1.5 and 14.5 ± 1.5, respectively). Beating waves propagated along the full length of the flagellum after sperm activation, whereas waves were restricted to the proximal section during the latter motility period. Wave amplitude significantly decreased at 45 s after sperm activation, but wavelength did not differ. The present study showed associations
among sperm morphology, ATP content, flagellar wave parameters, and sperm velocity, which could be used in comparative spermatology.
Résumé : Le spermatozoı̈de du flétan atlantique (Hippoglossus hippoglossus (L., 1758)) est uniflagellé, sans acrosome et
différencié en tête, corps médian et flagelle. Il y a deux à cinq mitochondries dans le corps médian, ainsi que des centrioles proximaux et distaux. Le flagelle consiste en 9 + 2 microtubules entourés d’une membrane plasmatique qui se prolonge
dans la partie proximale du flagelle à cause de la présence de vacuoles. Après l’activation des spermatozoı̈des dans l’eau
de mer, la motilité et la vitesse des spermatozoı̈des diminuent respectivement de 98,4 % ± 3,4 % et de 170,3 ± 8,9 mms–1
à 15 s après l’activation à 4,8 % ± 4,7 % et 9,2 ± 8,9 mms–1 à 120 s après l’activation. Le contenu en ATP (nmolL–1
ATP par 108 spermatozoı̈des) diminue significativement 60 s après l’activation (5,9 ± 1,5) par comparaison à 0 et 30 s
après l’activation (respectivement 14,9 ± 1,5 et 14,5 ± 1,5). Les ondulations de battement se propagent sur toute la longueur du flagelle après l’activation, mais sont restreintes à la partie proximale durant la période de motilité subséquente.
L’amplitude des ondulations diminue significativement à 45 s après l’activation, mais leur longueur d’onde ne change pas.
Notre étude met en lumière des associations entre la morphologie des spermatozoı̈des, leur contenu en ATP, les variables
ondulatoires des flagelles et la vitesse des spermatozoı̈des qui pourraient servir en spermatologie comparée.
[Traduit par la Rédaction]
Introduction
Morphology, motility, and seminal plasma composition
are considered major elements in the study of comparative
fish spermatology (Jamieson 1991; Ishijima et al. 1998;
Alavi et al. 2008a). Fish spermatozoa are differentiated into
a head, a midpiece, and a flagellum with the typical 9 + 2
pairs of microtubules (Jamieson 1991; Lahnsteiner and Patzner 2008). Spermatozoa of most fish species are immotile in
the testis or sperm duct prior to spawning (Morisawa and
Suzuki 1980). Activation of sperm occurs after release from
the genital papilla into an aquatic environment (Billard et al.
1995). The mechanism of sperm motility initiation differs
between freshwater and marine fishes with the former re-
Received 30 June 2010. Accepted 21 December 2010. Published on the NRC Research Press Web site at cjz.nrc.ca on 12 February 2011.
S.M.H. Alavi1 and A. Hatef.2 South Bohemia Research Center of Aquaculture and Biodiversity of Hydrocenoses, Faculty of Fisheries
and Protection of Waters, University of South Bohemia in České Budějovice, 389 25 Vodňany, Czech Republic.
I.A.E. Butts.2 Fisheries and Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, NB E5B 2L9, Canada;
Department of Biology, University of New Brunswick (Saint John), Ganong Hall, P.O. Box 5050, Saint John, NB E2L 4L5, Canada.
M. Mommens and I. Babiak.3 Reproductive Biology Research Group, Faculty of Biosciences and Aquaculture, University of Nordland,
Bodø, 8049, Norway.
E.A. Trippel. Fisheries and Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, NB E5B 2L9, Canada.
M.K. Litvak. Department of Biology, Mount Allison University, 63B York Street, Sackville, NB E4L 1G7, Canada.
1Corresponding
author (e-mail: [email protected]).
authors contributed equally to this work as second author.
3Corresponding author (e-mail: [email protected]).
2Both
Can. J. Zool. 89: 219–228 (2011)
doi:10.1139/Z10-113
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220
quiring a hypo-osmotic and the latter a hyper-osmotic signal
(Morisawa et al. 1999; Morisawa 2008). Duration of sperm
motility varies widely among species, although generally it
is briefer in freshwater than marine species (Trippel 2003;
Cosson 2010).
Seminal plasma is a secretory product of various reproductive accessory organs and tissues, and its primary function is to nourish sperm prior to release into the aquatic
environment (Alavi and Cosson 2006; Ciereszko 2008).
Seminal plasma also plays a crucial role in the maturation
and protection of sperm cells (Ciereszko 2008). Its composition varies among different groups of fishes and exhibits
intra- and inter-species variation (Alavi et al. 2008a; Butts
et al. 2010a).
Motility of spermatozoa depends on the energy released
by hydrolysis of adenosine-5’-triphosphate (ATP) by dynein
ATPase to produce flagellum beating (Gibbons 1981). Once
sperm are activated, motility and velocity decrease rapidly
(see review by Alavi and Cosson 2006; Cosson 2010) owing
to the depletion of ATP content (Perchec et al. 1995; Butts
et al. 2010b). This phenomenon leads to changes in beating
frequency of flagellum, wavelength, wave amplitude, and
number of propagating waves (Ishijima et al. 1998; Cosson
et al. 2008; Alavi et al. 2009a, 2009b; Cosson 2010), which
determine the sperm velocity (Alavi et al. 2009a; Cosson
2010). Additionally, size and shape of spermatozoa have
been associated with velocity and fertilization ability (Vladić
et al. 2002; Burness et al. 2005). As a result, quantifying
sperm velocity, beating parameters of flagellum, and ATP
content along with morphology of sperm are key components in gaining a better understanding of biophysical features of sperm motility that will ultimately provide us with
a more comprehensive knowledge of comparative spermatology.
Atlantic halibut (Hippoglossus hippoglossus (L., 1758))
(Pleuronectidae, Pleuronectiformes) is the world’s largest
flatfish. It is sought after by commercial fishers and its high
market value makes it a highly prized candidate for coldwater mariculture (Babiak et al. 2006; Mommens et al.
2008). In Norwegian coastal waters, halibut spawn from January to April at depths of 300–700 m within a narrow range
of temperatures (5–7 8C) and salinity (34.5–34.9 ppt) (Haug
1990). Despite its importance, only a limited amount of research has been conducted on halibut reproductive biology
with a large focus on female attributes (Norberg et al. 1991;
Mazorra et al. 2003).
Morphology of sperm has been recently reviewed in the
family Pleuronectidae (Jamieson 2009), but no information
is available on the Atlantic halibut. In halibut like in many
other teleosts (see review by Alavi et al. 2008a), sperm
quality differs throughout the spawning season (Babiak et
al. 2006). Moreover, a positive correlation exists between
spermatozoa concentration and osmolality of the seminal
plasma (Mommens et al. 2008) with both undergoing seasonal increases, which co-occur with declines in sperm motility, velocity, and fertilizing ability (Babiak et al. 2006).
Spermatozoa exhibit motility within a range of osmolality
of 400–1100 mosmolkg–1 (Billard et al. 1993). The initial
beat frequency is between 45 and 50 Hz at 15 s after sperm
activation and decreases to 10 Hz at 60–80 s after sperm activation (Billard et al. 1993).
Can. J. Zool. Vol. 89, 2011
Specific objectives of the present research were to investigate sperm morphology and ultrastructure, seminal plasma
composition, sperm ATP content, and dynamics of sperm
motility in Atlantic halibut. Wave parameters of flagella
were also analyzed to describe in detail the motility patterns
of spermatozoa.
Materials and methods
Broodstock husbandry and semen collection
Semen samples from 10 Atlantic halibut broodstock were
collected at the University of Nordland, Bodø, Norway, in
March 2009. Broodstock consisted of 6-year-old farmed
males held in 160 m3 tanks and fed once a week with Fish
Breed-M (INVE Aquaculture NV, Dendermonde, Belgium).
At time of sampling, males were held under a 19 h dark :
5 h light photoperiod regime (1 month advanced compared
with the natural photoperiod) at 5.5 ± 0.2 8C (mean ± SD),
33.5 ± 0.2 ppt (mean ± SD) salinity, and 86.5% ± 3.3%
(mean ± SD) oxygen saturation. Males were first placed on
a wet table; sperm was collected without anesthesia (Babiak
et al. 2006). The initial ejaculate was discarded and the external urogenital pore was wiped dry with paper towel to
avoid contamination from seawater, urine, and feces. Each
semen sample was obtained by applying slight abdominal
pressure and was collected in a syringe (5 mL) and placed
on crushed ice until processed (within 1–3 h).
Sperm morphology
Sperm from four males were fixed in Karnoysky’s solution containing 5% glutaraldehyde and 4% paraformaldehyde and kept at 4 8C until transfer to the South Bohemia
Research Center of Aquaculture and Biodiversity of Hydrocenoses in Vodňany and to the Electron Microscopy Laboratory in České Budějovice, Czech Republic. Samples were
postfixed and washed repeatedly for 2 h in 4% osmium tetroxide at 4 8C and dehydrated through an acetone series.
Samples for scanning electron microscopy were dehydrated
in a critical point dryer pelco CPD 2 (Ted Pella, Inc., Redding, California, USA). Sperm samples were coated with gold
under vacuum with a scanning electron microscopy Coating
Unit E5100 (Polaron Equipment Ltd., Hertfordshire, England) and observed using a JSM 6300 (JEOL Ltd., Akishima, Tokyo, Japan). Samples for transmission electron
microscopy were embedded in resin (Polybed 812). A series
of ultrathin sections were cut using a Leica UCT ultramicrotome (Leica Mikrosysteme Gmbh, Vienna, Austria) and double-stained with uranyl acetate and lead citrate. Samples
were viewed in a transmission electron microscope JEOL
1010 (JEOL Ltd., Tokyo, Japan) operated at 80 kV. Micrographs were evaluated using the Olympus MicroImage software (version 4.0.1. for Windows) to measure sperm
morphological parameters.
Seminal plasma composition and osmolality
Seminal plasma composition and osmolality of 10 males
were measured. Semen samples were centrifuged for 3 min
at 7000 rpm. The supernatant (seminal plasma) was then pipetted into an Eppendorf tube (2 mL) and stored frozen at
–80 8C until analysis. Osmolality of the seminal plasma
was measured in duplicate with a Vapour Pressure OsmomPublished by NRC Research Press
Alavi et al.
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eter (model 5520; Wescor, Inc., Logan, Utah, USA). Chloride, potassium, and sodium ions were measured using Ion
Selective Electrodes, ISE, (Bayer Healthcare, New York,
New York, USA) by indirect simultaneous measurement.
Calcium concentration was measured by flame photometry.
Ionic composition was measured once per sample.
Spermatozoon concentration
Spermatozoon concentration of 10 males was counted
under a light microscope, using a haemocytometer, following the method of Alavi et al. (2009a). In brief, semen was
diluted two times, each 100-fold with 0.7% NaCl (final dilution rate is 10 000). Ten microlitres of diluted semen was
placed onto a haemocytometer, covered with a cover slip,
and left for 10 min to allow sperm sedimentation before
16 cells (0.1 mm depth and 0.2 mm 0.2 mm square)
were counted.
221
distance between two successive peaks within one sine
wave. It should be noted that sperm motility of Atlantic halibut is symmetric (close to a true sine-wave shape); therefore, the use of peak amplitude and wavelength act as
unambiguous and reliable measurements.
Data analysis
All data were analysed using SAS version 9.1 (SAS Institute Inc., Cary, North Carolina, USA) and reported as
means ± SE unless otherwise indicated. Residuals were
tested for normality (Shapiro–Wilks test; PROC UNIVARIATE; SAS Institute Inc. 2003) and homogeneity of variance
(plot of residuals vs. predicted values; PROC GPLOT; SAS
Institute Inc. 2003). Data were transformed to meet the assumptions of normality and homoscedasticity when necessary. Alpha was set at 0.05 for main effects. Changes in
sperm motility, velocity, ATP, wavelength, and wave amplitude were analyzed using mixed-model repeated-measures
ANOVAs (PROC MIXED; SAS Institute Inc. 2003). The
Kenward–Roger and Satterthwaite procedures were used to
approximate the denominator degrees of freedom for unbalanced and balanced data, respectively (Spilke et al. 2005).
The repeated statement (repeated time after sperm
activation / subject = male) was used to model the covariance structure within subjects. Three covariance structures
were modeled: compound symmetric (type = cs), autoregressive order one (type = ar(1)), and ‘‘unstructured’’ (type =
un). Akaike’s (AIC) and Bayesian (BIC) information
model-fit criteria were used to assist in final model inference determination (Littell et al. 1996). Treatment means
were contrasted using the least-squares means method
(LSMEANS/CL adjust = TUKEY, PROC MIXED; SAS Institute Inc. 2003).
Linear regressions were run to determine the relationship
between flagellum wave parameters and sperm velocity
throughout the time course of sperm activity (PROC REG;
SAS Institute Inc. 2003).
ATP content
ATP content of five males was determined using the bioluminescence method described by Jeulin and Soufir (1992)
and Perchec et al. (1995). Sperm were activated in seawater
(semen:seawater ratio was 1:100) at room temperature (18–
21 8C). At 0, 30, 60, 90, and 120 s after sperm activation,
500 mL of this suspension was diluted in 5 mL of boiling
medium containing HEPES buffer 25 mmolL–1 HEPES,
10 mmolL–1 magnesium acetate, 2 mmolL–1 EDTA,
3 mmolL–1 sodium azide, pH 7.75, and left for 2 min at
98–100 8C. The sperm suspension was then centrifuged for
15 min at 14 000 rev/min (16 000 g). The supernatant was
removed and stored at –20 8C until analysis. The procedure
for ATP determination of sperm involved the addition of purified luciferin–luciferase kit by Bioluminescence Assay Kit
CLS II (Roche Diagnostics GmbH, Basel, Switzerland). Luminescence was recorded with a multifunctional microplate
reader infinite M200 (TECAN, Mannedorf, Switzerland).
ATP content of each sperm sample was calculated using the
standard method provided in the kit manual and reported as
nmolL–1 ATP per 108 spermatozoa.
Results
Sperm motility and analyses of flagellum wave
parameters
We examined changes in sperm motility, velocity, and
swimming behaviour over time in seawater of 10 males at
room temperature (18–21 8C). In all observations, BSA was
added at 0.1% (m/v) to prevent sperm from sticking to the
glass slides. For activation, sperm were first diluted in an
immobilizing solution (33% seawater) (semen:seawater ratio
was 1:10) and 0.1–0.2 mL of this suspension was then mixed
with 49 mL of seawater. Motility was recorded immediately
after activation using a 3 CCD video camera (SONY SSCDC50AP; SONY Corp., Tokyo, Japan) mounted on a microscope (Leica Laborlux K, Optomedic AS, Lysaker, Norway)
under stroboscopic light. Sperm motility, velocity, and flagellum wave parameters (wavelength and wave amplitude)
were analyzed on successive video frames using a micro-image analyzer (Olympus Micro Image version 4.0.1 for Windows) following Alavi et al. (2009b). Mean velocity was
based on motile spermatozoa only. Wave length was measured as the distance between repeating units of a propagating wave. Peak-to-peak amplitude was measured as the
Sperm morphology
Spermatozoa of Atlantic halibut are uniflagellated, lack an
acrosome, and have clearly differentiated spherical heads
(length along the longitudinal axis = 1.43 ± 0.01 mm), midpieces (length = 0.44 ± 0.01 mm), and flagella (length =
29.69 ± 0.17 mm) (Table 1, Fig. 1a). They are symmetrical
about its longitudinal axis (Figs. 1b, 1c). The head contains
a nucleus having an invagination called the nuclear notch,
where the centriolar complex is located (Fig. 1c). The proximal centriole is inclined to the distal centriole at an angle
of 1208. Centrioles consist of nine peripheral triplets of microtubules (Fig. 1d). The midpiece contains two to five mitochondria (Figs. 1c, 1d). The nucleus and midpiece are
surrounded by vacuolated cytoplasm (Figs. 1c, 1d). The
flagellum consists of 9 + 2 microtubules, which are constructed of nine outer doublet microtubules and a single central pair. Outer and inner dynein arms and a radial spoke are
present, which are involved in flagellum motility. It appears
that the axoneme is surrounded by vacuolated cytoplasm,
which leads to extension of the flagellum; mostly at the
proximal (close to head) compared with distal part
Published by NRC Research Press
222
Can. J. Zool. Vol. 89, 2011
Table 1. Morphological characteristics of sperm of Atlantic halibut (Hippoglossus hippoglossus) (n = 4).
Can. J. Zool. Downloaded from www.nrcresearchpress.com by 24.57.120.143 on 05/16/11
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Parameter
Head
Length (mm)
Width (mm)
Anterior midpiece length (mm)
Posterior midpiece length (mm)
Midpiece length (mm)
Flagellar length (mm)
Total length (mm)
Minimum
Maximum
Mean
SE
0.92
1.05
0.52
0.28
0.19
20.35
2.07
2.01
2.21
1.58
1.16
0.89
41.46
43.54
1.43
1.55
1.04
0.61
0.44
29.69
31.30
0.01
0.01
0.01
0.01
0.01
0.27
0.28
Fig. 1. Spermatozoa of Atlantic halibut (Hippoglossus hippoglossus). (a) Scanning electron micrograph (SEM) shows the head, midpiece,
and flagellum of sperm. (b) SEM showing the symmetrical pattern position of sperm. (c) Transmission electron micrograph (TEM) showing
the longitudinal section of the head region, midpiece, and flagellum. (d) TEM showing the cross section of the midpiece. N, head; M, midpiece; F, flagellum; nn, nuclear notch; DC, distal centriole; PC, proximal centriole; Mt, mitochondria.
(Figs. 1a, 2a, 2b). Lateral flagellar fins may be absent in Atlantic halibut.
2.6 ± 0.4 mgL–1. Osmolality of the seminal plasma was
362.2 ± 4.4 mosmolkg–1.
Seminal plasma composition and osmolality
The dominant ions in the seminal plasma were Na+
(178.5 ± 2.0 mmolL–1) and Cl– (165.4 ± 1.6 mmolL–1).
The concentrations of K+, Mg2+, and Ca2+ were 2.7 ± 0.6,
1.5 ± 0.5, and 1.3 ± 0.0 mmolL–1, respectively. Concentrations of glucose, cholesterol, and lactate were 0.1 ± 0.0,
0.4 ± 0.1, and 0.1 ± 0.0 mmolL–1, respectively, whereas
the concentration of total proteinin the seminal plasma was
Spermatozoa concentration
Spermatozoa concentration ranged from 11.5 109 to
30.4 109 spermmL–1, with a mean concentration of
22.4 109 ± 3.3 109 spermmL–1.
Sperm motility and ATP content
After triggering the initiation of sperm motility in seawater, sperm motility (F[7,50] = 56.9, p < 0.0001) and velocPublished by NRC Research Press
Alavi et al.
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Fig. 2. Transmission electron micrograph showing the cross section of the axoneme of sperm of Atlantic halibut (Hippoglossus hippoglossus) at proximal (a) and distal (b) parts of the flagellum.
ity (F[7,33.3] = 117.8, p < 0.0001) decreased over time
(Figs. 3a, 3b). Sperm motility was 98.4% ± 3.4% at 15 s
after sperm activation and decreased to 4.8% ± 4.7% at
120 s after sperm activation (Fig. 3a). Sperm velocity decreased from 170.3 ± 8.9 mms–1 at 15 s to 9.2 ±
8.9 mms–1 at 120 s after sperm activation. ATP content of
nonactivated sperm was not different from that of sperm at
30 s after activation, but a significant decrease was observed
at 60 s after sperm activation (F[6,15.7] = 15.3, p < 0.0001;
Fig. 4).
Analyses of flagellum wave parameters
Immediately after the triggering of sperm motility in seawater, beating waves propagated along the full length of a
flagellum, whereas waves at later periods of the motility
phase were restricted to the proximal section of the flagellum (close to head). Waves were completely absent at the
end of the motility phase (Figs. 5a–5e). Wavelength ranged
from 11.3 ± 0.4 mm at 90 s after sperm activation to 12.4 ±
0.3 mm at 30 s after sperm activation, with no significant
change during the activation timeline (F[5,36.4] = 1.3, p >
0.05; Fig. 6a). Wave amplitude significantly decreased at
30 s after sperm activation (F[5,27.1] = 92.5, p < 0.0001;
Fig. 6b). Significant relationships were observed between
wavelength and sperm velocity (F[4,4.8] = 20.0, p < 0.01,
Fig. 7a) and between wave amplitude and sperm velocity
(F[4,11.5] = 134.4, p < 0.0001; Fig. 7b) during sperm motility.
Discussion
Sperm morphology of Atlantic halibut is similar to that of
most freshwater and marine fishes in that spermatozoa comprised a head, midpiece, and flagellum with a 9 + 2 structure of microtubules and lack an acrosome (Lahnsteiner and
Patzner 2008; Jamieson 2009). Previous studies demonstrated interspecies and family differences for head shape,
number of mitochondria, location and positions of proximal
and distal centrioles, presence of a fin structure, and dimensions of head, midpiece, and flagellum. For example, spermatozoa of starry flounder (Platichthys stellatus (Pallas,
1788)), marbled flounder (Pseudopleuronectes yokohamae
(Günther, 1877)), little mouth flounder (Pseudopleuronectes
herzensteini (Jordan and Snyder, 1901)), Mediterranean
horse mackerel (Trachurus mediterraneus (Steindachner,
1868)), boga (Boops boops (L., 1758)), and white seabream
(Diplodus sargus (L., 1758)) have a head of ovoid or ellipsoidal shape, but spermatozoa of dusky spinefoot (Siganus
fuscescens (Houttuyn, 1782)), Atlantic halibut (present
study), olive flounder (Paralichthys olivaceus (Temminck
and Schlegel, 1846)), red seabream (Pagrus major (Temminck and Schlegel, 1843)), and goldline seabream (Rhabdosargus sarba (Forsskål, 1775)) have a spherical-shaped
head (Lahnsteiner and Patzner 1998; Chang and Chang
2002; Gwo et al. 2004a, 2004b). Spermatozoa of
bluntsnouted mullet (Mullus barbatus L., 1758) and Japanese goatfish (Parupeneus spilurus) have long heads that
are flattened on one side (Lahnsteiner and Patzner 1998;
Gwo et al. 2004b). The number of mitochondria is eight in
Pseudopleuronectes yokohamae; seven in S. fuscescens,
Platichthys stellatus, and Pseudopleuronectes herzensteini;
six in Parupeneus spilurus and Paralichthys olivaceus; five
in Atlantic halibut (present study) and M. barbatus; four in
T. mediterraneus; two in R. sarba; and one in Pagrus major,
B. boops, and D. sargus (Lahnsteiner and Patzner 1998;
Gwo et al. 2004a, 2004b). Flagellum length in spermatozoa
of Atlantic halibut (29.69 mm) is shorter than that of M. barbatus, T. mediterraneus, B. boops, and D. sargus (40–
45 mm; Lahnsteiner and Patzner 1998). Paired lateral fins
along the flagellum are observed in M. barbatus, but a single-side fin was reported in B. boops and D. sargus (Lahnsteiner and Patzner 1998). In the present study, no fin was
observed in sperm of Atlantic halibut, similar to those of
Pagrus major, R. sabra, Parupeneus spilurus, S. fuscescens,
and T. mediterraneus, but the axoneme was surrounded by a
wide layer of vacuolated cytoplasm similar to those of Paralichthys olivaceus, Platichthys stellatus, Pseudopleuronectes yokohamae, and Pseudopleuronectes herzensteini
(Gwo et al. 2004a, 2004b; Chang and Chang 2002). In the
present study, we also observed vacuolated cytoplasm
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Fig. 3. (a) Motility and (b) velocity of sperm of Atlantic halibut
(Hippoglossus hippoglossus) after activation in seawater. Sperm
motility (y = –15.01x + 122.23, r = 0.98, n = 10, p < 0.0001) and
sperm velocity (y = –20.17x + 175.76, r = 0.99, n = 10, p < 0.0001)
decrease after activation. Values with different letters are significantly different (p < 0.05).
around the nucleus and in the midpiece. These sperm head
differences might be related to the morphology of the micropyle, which the sperm penetrates to achieve egg fertilization
(Hatef et al. 2011). In addition, the differences in dimensions of morphological parameters of sperm could partly explain the observed differences in sperm motility dynamics
and beating parameters of flagellum (Alavi et al. 2009b).
These differences in morphology and motility of sperm
could be of use for fish taxonomy, and help to explain reproductive ecology and mating behaviour, as well as spermatozoa swimming performance (Ishijima et al. 1998;
Lahnsteiner and Patzner 2008).
Spermatozoon concentration in the present study (22.4 109 ± 3.3 109 spermmL–1) was higher than previously
reported for Atlantic halibut (15 109 to 18 109
spermmL–1, Babiak et al. 2006; 11.5 109 to 21.1 109
spermmL–1, Mommens et al. 2008). In each of these studies, samples from Atlantic halibut were collected in March.
The observed differences might therefore be related to fish
origin (wild or cultivated), donor age, and broodfish rearing
Can. J. Zool. Vol. 89, 2011
Fig. 4. ATP content of nonactivated and activated sperm of Atlantic halibut (Hippoglossus hippoglossus) when motility was triggered
in seawater. ATP content decreased after activation (y = –3.55x +
18.89, r = 0.93, n = 5, p < 0.0001). Values with different letters are
significantly different (p < 0.05).
conditions (Alavi et al. 2008a; Butts et al. 2010a). Osmolality of the seminal plasma (362.2 ± 4.4 mosmolkg–1) was
similar to previous studies on halibut (371–384
mosmolkg–1, Babiak et al. 2006; 361–385 mosmolkg–1,
Mommens et al. 2008) and for other marine fish such as
cod (Gadus morhua L., 1758) (355–414 mosmolkg–1, Litvak and Trippel 1998; Butts et al. 2010a) and turbot (Psetta
maxima (L., 1758)) (317 mosmolkg–1, Dreanno et al.
1999b). Osmolality of the seminal plasma in freshwater species is usually <300 mosmolkg–1 (Alavi et al. 2008a). Ionic
composition of the seminal plasma was similar to other marine species such as Psetta maxima (Dreanno et al. 1999b)
and G. morhua (Butts et al. 2010a). In marine species, Na+
and Cl– are the predominant ions and K+ concentration is
very low (<5 mmolL–1) compared with freshwater species,
which have higher K+ concentrations; 30–80 mmolL–1 in
cyprinids and 20–65 mmolL–1 in salmonids (Alavi et al.
2008a). Protein concentration in the seminal plasma (2.60 ±
0.36 mgL–1) from our study was lower than 6.4–
19.4 mgmL–1 reported by Mommens et al. (2008). The differences in ionic and organic compositions and osmolality of
the seminal plasma as mentioned above may be a result of
differences in sperm-duct secretory activity, spawning induction, broodfish origin, rearing condition, and other factors that may influence hormonal regulation of spermiation
(Ciereszko 2008; Alavi et al. 2008a; Butts et al. 2010a).
In this study, the ATP content of sperm from Atlantic halibut was 14.9 ± 1.6 nmolL–1 per 108 spermatozoa before activation, which is close to that in the sperm of the common
carp (Cyprinus carpio L., 1758) (15.2 ± 2.4 nmolL–1 per
108 spermatozoa, Perchec et al. 1995) and slightly higher
than in the sperm of sea bass (Dicentrarchus labrax (L.,
1758)) (11.9 ± 1.1 nmolL–1 per 108 spermatozoa, Dreanno
et al. 1999a) and sheatfish (Silurus glanis L., 1758)
(13.0 nmolL–1 per 108 spermatozoa, Billard et al. 1997).
Higher concentrations of ATP have been reported in Psetta
maxima (26.0 ± 3.8 nmolL–1 per 108 spermatozoa, Dreanno
et al. 1999b). Sperm motility depends on dynein ATPases,
which hydrolyse ATP to produce flagellum beating (Gibbons 1981). Motility is dependent both on ATP stored prior
to ejaculation (Christen et al. 1987) and on ATP synthesized
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Alavi et al.
225
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Fig. 5. Motility of sperm of Atlantic halibut (Hippoglossus hippoglossus) in seawater after (a) 15 s, (b) 30 s, (c) 60 s, (d) 90 s, and (e) 120 s
after sperm activation.
Fig. 6. (a) Wave length (y = –0.24x + 12.8, r = 0.95, n = 5, p <
0.001) and (b) wave amplitude (y = –0.59x + 5.79, r = 0.99, n = 5,
p < 0.001) during the motility of sperm of Atlantic halibut (Hippoglossus hippoglossus). Wave length did not show significant change
during activation timeline. In the case of wave amplitude, values
with different letters are significantly different (p < 0.05).
during the motility phase (Lahnsteiner et al. 1999). It was
shown that the mitochondrial oxidative phosphorylation is
unable to compensate for the ATP hydrolysis required to
sustain motility (Perchec et al. 1995). Therefore, decreasing
ATP content and accumulation of adenosine-5’-diphosphate
(ADP) could explain the brief period of sperm motility
(Dreanno et al. 1999a), which contributes to a decrease in
flagellum beat frequency and subsequently sperm velocity
(Omoto 1991; Cosson 2010). Level of decline of ATP after
sperm activation differs among species. For example, ATP
content significantly decreased at 10 s after sperm activation
in Dicentrarchus labrax (Dreanno et al. 1999a), but at 30 s
Fig. 7. Relationships between (a) wave length and sperm velocity
(y = 103.49x – 1129.60, r = 0.91, p < 0.01) and (b) wave amplitude
and sperm velocity (y = 41.65x – 50.65, r = 0.98, p < 0.0001) during the motility of sperm of Atlantic halibut (Hippoglossus hippoglossus).
after activation in bluegill sunfish (Lepomis macrochirus Rafinesque, 1819) (Burness et al. 2005), G. morhua (Butts et
al. 2010b), and Eurasian perch (Perca fluviatilis L., 1758)
(A. Hatef and S.M.H. Alavi, unpublished data), and after 1
or 2 min in S. glanis (Billard et al. 1997) and C. carpio
(Perchec et al. 1995). It has been well documented that decreases in sperm motility and velocity result from depletion
of ATP content (Perchec et al. 1995; Dreanno et al. 1999a;
Cosson et al. 2008). Initial sperm motility depends on maturation of sperm, which is regulated by 17,20b-dihydroxypregn-4-en-3-one, or in certain species, 17,20b,21trihydroxy-pregn-4-en-3-one (see review by Alavi et al.
2008a; Scott et al. 2010).
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226
Differences in sperm velocity among species may result
from variations in head shape, which may influence the
hydrodynamic properties of spermatozoa, the number of mitochondria, which determines the energy supply (initial ATP
content), and the size and shape of lateral ribbons (fins)
(Lahnsteiner and Patzner 1998; Alavi et al. 2009b; Cosson
2010). Sperm velocity in Atlantic halibut (152 mms–1) was
similar to that in M. barbatus (161 mms–1) with paired lateral fins, but higher than in other species that lack lateral
fins along the flagellum such as T. mediterraneus
(119 mms–1). This might be due to the presence of vacuoles
around the axoneme, which leads to extension of the flagellum. In B. boops and in D. sargus with an unpaired lateral
fin, sperm velocity was 125–127 mms–1 (Lahnsteiner and
Patzner 1998). Similar observations have been reported in
freshwater species, e.g., higher sperm velocities were noted
in northern pike (Esox lucius L., 1758) (175–185 mms–1,
Alavi et al. 2009b), which is characterized by an unpaired
lateral fin, and in sterlet sturgeon (Acipenser ruthenus L.,
1758) (160–180 mms–1, Alavi et al. 2008b) with paired lateral fins compared with that of the common barbel (Barbus
barbus (L., 1758)) (95–105 mms–1, Alavi et al. 2009a) or
Perca fluviatilis (105–125 mms–1, Alavi et al. 2007), which
have no fin structure. It is possible that lateral fins or presence of vacuoles in flagellar structure may increase the force
of beating and aid in self-propulsion (Lahnsteiner and Patzner 1998; Alavi et al. 2009b).
Similar to other fish species (Cosson et al. 2008, 2010;
Alavi et al. 2009a, 2009b), waves appear along the full
length of the sperm flagellum of Atlantic halibut after initiation of sperm motility. The waves were observed only close
to the head at the later period of motility and became absent
at the end of motility. The activity of inner dynein arms
generate flagellum bends, determine the size and shape of
the waveform, whereas the outer dynein arms add power
and increase beat frequency (Brokaw and Kamiya 1987;
Mizuno et al. 2009). Studies on fish sperm revealed that
wavelength and amplitude usually decrease after activation
(Alavi et al. 2008b, 2009b). Initial wavelengths were 15.5,
14.7, 12.3, and 12.0 mm in A. ruthenus, E. lucius, Atlantic
halibut, and European hake (Merluccius merluccius (L., ))
at 10–15 s after sperm activation, respectively (Alavi et al.
2008b, 2009b; Cosson et al. 2010; present study). Similar to
recent published data on sperm of M. merluccius (Cosson et
al. 2010), we did not find a significant change in sperm
wavelength over time in the present study. However, we did
see a significant decrease in wave amplitude. The reported
decrease from 5.1 to 3.2 mm for the Atlantic halibut in our
study is very similar to that in A. ruthenus (from 7.5 to
7.0 mm, Alavi et al. 2008b), E. lucius (from 5.3 to 2.6 mm,
Alavi et al. 2009b), and M. merluccius (from 4 to 1.5 mm,
Cosson et al. 2010). Sperm velocity was correlated with
flagellum wave length and wave amplitude during sperm
movement. These data suggest that the activity of dynein
arms corresponds with flagellum beating features. Similar
observations have been reported in E. lucius (Alavi et al.
2009b), A. ruthenus (Alavi et al. 2008b), and marine species
such as G. morhua, Psetta maxima, and M. merluccius
(Cosson et al. 2008, 2010; Cosson 2010).
In conclusion, spermatozoa of Atlantic halibut exhibited
similar morphological structure to the spermatozoa of the
Can. J. Zool. Vol. 89, 2011
other fish species, but differences were observed in terms of
the morphological dimensions and ultrastructural features
such as fin structure along the flagellum and number of mitochondria. These parameters may lead to differences in
sperm motility characteristics such as velocity and the initial
ATP store of sperm among species. The present study also
showed significant correlations between sperm velocity and
wave parameters of the flagellum. However, further studies
(i.e., meta-analysis) are required to answer whether the interspecies differences in sperm velocity or wave parameters
of the flagellum are a function of the flagellum length, the
head shape, and the extension of the flagellum, as well dynein ATPase activity, the latter regulating wave generation
and their shape and size. Taken together, sperm morphology
and motility characteristics could be used as a tool for comparative spermatology in fish taxonomy.
Acknowledgements
We thank Teshome Bizuayehu, Carlos Frederico Ceccon
Lanes, Michal Duc, Heidi Ludviksen, and Tormod Skaalsvik
for help with gamete collection. This work has been supported
by CENAKVA CZ.1.05/2.1.00/01.0024, MSM6007665809,
GA523/09/1793, IAA608030801, GAJU 046/2010/Z, and
Research Council of Norway project 182653/V10. Co-operation
with I.A.E.B was made possible thanks to the Natural Sciences and Engineering Research Council of Canada
(I.A.E.B.; NSERC PGS D3), the John S. Little International Study Fellowship (I.A.E.B), Marguerite and Murray
Vaughan Graduate Fellowship (I.A.E.B), Genome Canada,
Genome Atlantic, and the Atlantic Canada Opportunities
Agency through the Atlantic Cod Genomics and Broodstock Development project.
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