Determination of Sperms ATP Content of Golden Grey Mullet (Mugil auratus) at Different Conditions
Aim of this study was determination of sperms ATP content of golden grey mullet in different time, temperature and extenders. Caspian Sea Mugilidea is one of the most important fish of the sea fishery which nowadays is the predominant catches fish of the mentioned sea. The ATP content of mentioned fish sperm is an important index of fish fertility determination of the samples ATP concentration was done by ultra sensitive bioluminescence method. ATP content of sperms were determined at two different sampling temperature (10-12 and 18-20°C) and two different keeping temperature (4°C and room temperature) for 6 h and also ATP content assayed until 10 days storage in the three extender types (glycerol, 0.7 and 0.65% salt solution). Results of the present study showed that, ATP content of sperms, collected at 10-12°C was 74.04±7.22%, in comparison to 18-20°C. The ATP content of sperms during 6 h keeping at 4°C and room temperature were 90.26±0.91% and 17.17±1.49%, respectively. Determination of sperms ATP content after 5 days keeping in glycerol, 0.7 and 0.65% salt solution revealed that glycerol or 0.65% salt solution is better extender than 0.7% salt solution. But sperms which were kept in mentioned extenders for 10 days showed that glycerol was better than salt solutions based on sperm ATP content saving. Results revealed that, in order to save sperms ATP content of golden grey mullet, sampling at 18-20°C and keeping in glycerol as extender is recommended.
Sperm is an important factor of reproduction, survival and stock duration.
Environmental variables, particularly nutrition, are ultimately important in
affecting sperm quality and reproductive timing (Carl et
al., 2001). Study of sperm by parameters like fertility, motility, rate
of motile sperms, motility speed, flagella beating frequency, ATP content, chemical
and biochemical factors, help us for distinction of sperm quality and control
the reproduction procedure of animals such as aquatic creatures. As a result
expanded investigations were done by different researchers on this cell. The
studies in relation to sperm ATP content of fish are slight, so any research
has been done on this subject and this matter could be a cause for an unsuccessful
artificially bred process of some fishes. On this issue more studies should
be done on the basis of determination of sperms ATP content of fishes.
Sperms swim using flagella beating, the energy for which is provided by the
hydrolysis of ATP (Cosson et al., 2000; Kim
et al., 2009). Motility is dependent both on ATP which is stored
prior and that synthesized during the motility phase (Burness
et al., 2004). In most externally fertilizing fish species, ATP synthesis
is unable to sustain the high rates of ATP hydrolysis which is required during
motility and as a result ATP levels rapidly decline (Yeates
et al., 2007). This decline in ATP is paralleled by decline in flagella
beat frequency and sperm swimming speed (Christen et al., 1987; Perchec
et al., 1998). There is considerable variation among species of fish
in the content of ATP used to support motility of sperms (Mansour
et al., 2003; Casselman, 2006). In this study, sperms ATP content
of golden grey mullet was determined in different condition, time and temperature.
MATERIALS AND METHODS
In this study, in 30th Oct 2005 some golden grey mullet were caught from Caspian
sea with water temperature 18-20°C and salinity 12-13 ppm. The fishes were
caught for repeating in storm and reduced weather temperature (in 6th Nov 2005)
which water temperature was 10-12°C and salinity 12-13 ppm. Ten males (didn't
spawning) were selected and made ready sample of them. They were approximately
25-30 cm length and 900-1000 g weight. After sampling, sperms were immediately
transferred to laboratory in a closed test tube, in ice box. In order to determine
ATP content of stored sperms in room temperature and 4°C for 6 h, amount
of sperm sample of each fish were sampling in 10-12°C, stored in 4°C
and room temperature. Also, for determination of ATP content of stored sperms
at cold in different extenders (glycerol 10% and glucose 0.3 M, 0.7 and 0.65%
salt solution) after 5 and 10 days, 1 volume of sperm with 3 volume of extender
were slowly mixed. Sperm samples mixing with glycerol and 0.65% salt solution
were stored at -15°C and samples mixing with 0.7% salt solution stored at
1°C. In order to determination of ATP content of fresh sampled sperms (in
18-20°C and 10-12°C sea water) and the kept one (in 4°C and room
temperature), firstly sperms were diluted 1:100 in HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, Molecular Formula: C8H18N2O4S)
buffer solution (25 mM HEPES, 10 mM magnesium chloride, 2 mM EDTA, pH = 7.7).
Then for membrane destruction and ATP exiting (Billard et
al., 1999), micro vials were boiled in water bath at 100°C for 1
min. After mixing 50 μL of kit (Biaffin Co, GmbH, Germany) with 50 μL
micro vial contain in special plate of luminescence measurement, mentioned signals
measured by Luminometer in room temperature after 10 min. Taking time more than
30 caused to fall signal and standard curve invalidation. In later stage, standard
curve drew on luminescence signals and ATP concentration and at the end ATP
content of each fish sperm sample expressed as nmol ATP/108 sperm. In order
to determination of ATP content of keep sperms in cold and different extenders
after 5 and 10 days, firstly, samples which were frozen in -15°C (samples
keep in glycerol and 0.65% salt solution) exited from freezer and thawed in
37°C for 30 sec (Long et al., 1993). The
stages and processes in this experiment were repeated again on the kept samples
which were in cold and different extenders, after 5 and 10 days (Yeates
et al., 2007).
In order to express the ATP content according to cell number, the number of
sperms in1 ml milt sampled from 10 golden grey mullet, counted by Neobar lam
and microscope (Table 1). The Result of the mean of counting
the number of mentioned sperms was 239x108±15x108.
IN this study in order to determine the amount of effect of sampling temperature
on ATP content of fish sperm, sampling was done in 2 temperature 10-12 and 18-20°C
(Fig. 1). ATP content of the sampled sperms in mentioned temperatures
measured by bioluminescence sensitive procedure. The Results of this determination
showed that, ATP content of sperms, collecting at 10-12°C was 74.04±7.22%,
in comparison to 18-20°C. The temperature in which the sperms were kept
had an effect on ATP content of the mentioned cells. Finding the suitable temperature
in keeping the sperms, they were kept in 4°C and room temperature for 6
h and then the amount of ATP content of them was measured. Result of this determination
showed that ATP content of sperms during 6 h keeping at 4°C and room temperature
were 90.26±0.91 and 17.17±1.49%, respectively. Usually for keeping
the sperm extenders is used (Fig. 2). The extenders cause
to duration and protection of sperms activity in the keeping period.
|| Results related to number of the sperms exiting in 1cc milt
||Comparison of the effect of temperature on ATP content in
sperm of golden grey mullet **Significant difference (p<0.01)
||Effect of temperature on keeping sperm of golden grey mullet
for 6 h. ***Significant difference (p<0.001)
||TP's percentage of sperms in different days in glycerol, 0.65
and 0.7% salt solutions. *Significant difference from zero day (p<0.05)
#Significant difference from zero day (p<0.001)
For determining the best extender, glycerol 10% and glucose 0.3 M, 0.7 and
0/65% salt solution were investigated. ATP content of sperms in mentioned solutions
were measured after which sperm was kept in 1 and -15°C for 5 and 10 days.
Result of this measurement revealed that glycerol or 0.65% salt solution is
better extender than 0.7% salt solution. Trend of ATP content changes in different
extenders during 10 days showed that glycerol was better than salt solutions
for sperm ATP survival (Fig. 3).
More researches in related to fish sperm have done in world which any one view
pointed special side. A lot of research have done all around the world in related
to fish sperm but each of them survey the subject from their own point of view
(and look at it from the special angle) (Casselman, 2006).
However, there is limited information in the case of the sperm ATP content of
golden grey mullet and effects of environmental, chemical and biochemical factors
on it. Up to now, there is no investigation in related to the sperm ATP content
of golden grey mullet (until hasnt done), or (no investigation had been
done yet on
) golden grey mullet is one of important species of fish fishery
which until now (to be unsuccessful) reproduction and nourishment of it remains
unsuccessful. Maybe can use from this way for induced the favorite conditions
in order to reproduction and nourishment this fish species and or in special
conditions for sperm keeping. May be this experiment and its findings could
smooth the path to induce the favorite conditions in order to reproduction and
nourishment this fish species. It might be useful for finding the best condition
in which the sperm must be keeping. Perchec et al.
(1998) showed in the absence of urine contamination, spermatozoa had ATP
content in the range of 8-9 nmol/108 sperm. In contrast, when milt was contaminated
with urine, the ATP content was 4-5 nmol/108 sperm. Indeed the low osmolality
of urine by an early activation cause to early degradation in sperm ATP content
and decrease in the quality of carp spermatozoa. So, in this study for prevention
of sperm quality decreasing, during of sampling, sperm contamination by urine
or any other pollutant be prevented. Burness et al.
(2004), for determination of ATP content, add the luciferin-luciferase (dissolved
in 100 mM glycine, 20 mM Mgso4, pH 7.4) to sample and measured bioluminescence
signals by using a 96-well Lmax Luminometer (Molecular Devices, Sunnyvale, CA,
USA). The ATP content of each sample calculated by using ATP standards and expressed
as pmol/108 sperm. De Baulny et al. (1999) measured
the ATP content by using bioluminescence (ATP bioluminescence assay, kit cls;
Boehringer-Mannheim) after extraction with a trichloroacetic acid 2%, EDTA 2mM
medium and expressed as nmol/108 sperm. Perchec et al.
(1998) measured the ATP content by bioluminescence. First the sperm lyses
of common carp by 1/100 dilution into boiling HEPES buffer and then with addition
luciferin-luciferase to sperm, luminescence signals measured using a Biocounter
M2010A Lumac/3M. Then the ATP content calculated using ATP standards and expressed
as nmol/108 sperm (Kim et al., 2009). In this
study for measurement the ATP content used bioluminescence procedure. First
the sperm sample mixed 1/100 into boiling HEPES buffer then 50 μL of it
with 50 μL of bioluminescence sensitive kit (Biaffin Co, GmbH, Germany)
mixed into special plate of luminescence measurement and luminescence signals
measured using Luminometer (Lumistar, BMG Labtech GmbH, 5|10-19 mol/well,
Germany). Then the ATP content of each sample calculated using ATP standards
and expressed as nmol/108 sperm. Results due to this study defined the appendix
The ATP content of sperms fresh sampled in 10-12°C, was 74.04±7.22%, ATP content of sperms fresh sampled in 18-20°C (sperm sampled in 18-20°C, 17.04±1.09 nmol/108 sperm, N=10, sperm sampled in 10-12°C, 12.21±0.9 nmol/108 sperm, N = 10, p>0.05).
The most sperm motility and its ability for insemination of egg is seen after
sampling. Sometimes in case of artificial insemination the sperm keeping is
necessary. Period of sperm motility is also affected by the keeping temperature
so that according to reports of Slominska (Slominska and
Jezierska, 2000), short term (15 min) keeping of sperm at 20°C shortened
their motility time and they also observed that the sperm motility of common
carp reduced by about 50% after 2 h keeping in 20°C. The results of research
of these 2 coworkers indicated the possibility of milt storage in refrigerator
(5°C) up to 24 h for common carp and 8 hours for grass carp and S. salar,
without considerable reduction of sperm quality (Yeates
et al., 2007). The number of sperm (Sander vitreus) used in
the experimental trials was kept constant, variation in sperm swimming speed
(at 10 s after activation) explained approximately 90% of the variation in a
male's fertilization success. These findings demonstrate that the variation
in sperm quality found in wild spawning populations has the potential to dramatically
influence male reproductive success (Casselman et al.,
The obtained results from this study indicated that ATP content of stored sperm in 4°C for 6 h was 90.26±0.91% ATP content of fresh sampled sperm in 10-12°C, that this difference was not significant (n = 10, p>0.05). The ATP content of sperm keep in laboratory for 6 h was 17.17±1.49% the ATP content of fresh sampled sperm in 10-12°C. Mentioned difference was significant (n = 10, p<0.001).
The ATP content of the kept sperms in glycerol at -15°C after 5 days, the ATP content of the kept sperms in salt solution 0.7% at 1°C after 5 days and The ATP content of the kept sperms in salt solution 0.65% at -15°C after 5 days respectively was 74.19±4.5, 13.65±2.81, 73.58±6.68% the ATP content of fresh sampled sperms in 10-12°C, that this difference was significant (n = 10, p<0.001), glycerol and salt solution 0.65% as compared with salt solution 0.7%, preserve the upper percent of ATP.
The ATP content of the kept sperms in glycerol at -15°C after 10 days, ATP content of the kept sperms in salt solution 0.7% at 1°C after 10 days and ATP content of the kept sperms in salt solution 0.65% at -15°C after 10 days respectively was 47.67±6.2, 0.69±0.06 and 1.05±0.12% ATP content of fresh sampled sperm in 10-12°C, that this difference was significant (n = 10, p<0.001) and glycerol as compared with salt solution 0.7 and 0.65% which preserve the upper percent of ATP.
Keeping the sperm of European catfish Silurus glanis by cryo preservation with
different cryo protect ants (Me2SO, DMA, glycerol, methanol and propylene
glycol) indicated that the best protection was given by DMA (10,15%), Me2SO
(15%). Calvi (Calvi and Maisse, 1998) in case of trout
Oncorhynchus mykiss reported that ATP level and sperm motility of this
fish considerably decreased after thawing. But De Baulny
et al. (1999) reported that European catfish sperm behave very differently
without decrease of ATP level (Stoltz and Neff, 2006).
European catfish frozen sperm with DMA result in a considerable increase of
ATP level of sperm. Because DMA causing to direct stimulation of ATP synthesis
that this increase of the ATP level during the process is probably a decisive
factor in the freezing resistance of European catfish sperm in the presence
of DMA. But obtained results from experiments of this study showed that the
best extender is glycerol 10% and glucose 0.3 M.
The ATP content of the kept sperms in glycerol in -15°C after 5 days and ATP content of the kept sperms in glycerol in -15°C after 10 days respectively was 74.19±4.5 and 47.67±6.2% ATP content of fresh sampled sperms in 10-12°C, that this difference was significant (n = 10, p<0.001).
The ATP content of the kept sperms in salt solution 0.7% in 1°C after 5 days and the ATP content of the kept sperms in salt solution 0.7% in 1°C after 10 days respectively was 13.65±2.81 and 0.69±0.06% the ATP content of fresh sampled sperms in 10-12°C, that this difference was significant (n = 10, p<0.001).
The ATP content of the kept sperms in salt solution 0.65% in -15°C after 5 days and the ATP content of the kept sperms in salt solution 0.65% in -15°C after 10 days, respectively was 73.58±6.68 and 1.05±0.12% the ATP content of fresh sampled sperms in 10-12°C, that this difference was significant (n = 10, p<0.001).
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