International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2011)

Effect of Sperm pH and Density on

Fertilization and Hatching Rates of Persian
Sturgeon (Acipenser persicus Borodin, 1897)
H. Dadras , H. Khara, s. Baradaran Noveiri E. shahkar
success in hatcheries. The aim of this study was finding the
correlations between two biomarkers of Persian sturgeon
sperm quality, including sperm density [34] and pH of sperm
[27] with the fertilization and hatching rates over an artificial
reproduction season.

Abstract This study was carried out during the artificial

reproduction season at the Shahid Beheshti Sturgeon Rearing and
Propagation Center and International Sturgeon Research Institute
(Rasht) during 2008-2009 breeding season. Eleven wild male
Acipenser persicus brood fish were used for this study. Each brood
fish was stripped after receiving hormonal injection and then sperm
samples were considered separately. Fertilization and hatching rates
of larvae were also determined. Analysis of the relationship between
sperm pH and density and indices of reproduction showed positive
and direct relationship between hatching rate and sperm pH (p <
0.05, r = 0.711), as well as between sperm pH and fertilization rates
(p < 0.05, r = 0.633). However negative relationship was observed
between sperm density and fertilization rates (p < 0.05, r = 0.640)
and between sperm density and hatching rates (p < .01, r = 0.745) .

II. MATERIALS AND METHODS

For this study 11 wild matured Acipenser persicus males
(Mean weight and length 12.43 1.93 kg and 135.91
14.28 cm respectively), caught from sturgeon fishing station
centers along Guilan Province at the southern Caspian Sea
during reproduction season 2008-2009 were studied. After
being transferred to breeding and reproduction complex in
Shahid Beheshti (Rasht,Iran). Spermiation was induced by
single intramuscular injection of LHRH-A2 at adose of 2-3
micrograms per kilogram of body weight with 2 ml saline
solution (% 0.6 NaCl). Semen samples were collected after 24
hours and transported immediately to fish sperm
cryopreservation laboratory (International sturgeon research
institute, Rasht) for further analyses. Sperm samples of each
brood stock were added to eggs at 10 ml of semen per kg eggs
(along with 100 ml of water per one ml of semen) and were
performed semi-dry method of fertilization. To determine the
fertilization rate, 300 eggs were selected, randomly removed
and fixed in formalin solution (one part 40% formalin mixed
with 9 parts water) and was observed by the binocular loop.
The fertilization rate was calculated through the following
relationship 3-4 hour after fertilization at 22-23 C . For this
purpose, 100-150 eggs were sampled randomly from each
incubator in the time of second mitosis division [25]:
0B

Keywords Fertilization Rate, Hatching Rate, Persian Sturgeon,

Sperm Density, Sperm pH.
I. INTRODUCTION
ERSIAN sturgeon (Acipenser persicus Borodin, 1897) is
considered as one of the most important Caspian
stuergeon species due to its high economic value. According
to live valuable resources of native and commercial species in
the Caspian Sea, its catching efforts during the last decade
have been decreased [32]. The purpose of sturgeon artificial
reproduction centers is releasing of sturgeon fingerlings into
the natural waters to compensate of this decline and enhancing
of fish catching efforts [35, 13]. Proper evaluation of sperm
quality characteristics is considered as a key factor during fish
reproductive activities [3]. Sperm density is a criterion and
main indicator to evaluate the sperm potential that will affect
the rate of fertilization [1, 31]. On the other hand pH will be
known as an important factor in sperm development [11] and
with other external factors appears a key role in the sperm
physiology [6]. pH may be considered as an important factor
leading to sperm activation in many fish species such as
Hippoglossus hippoglossus, Oncorhynchus mykiss, Polyodon
spathula [9,10, 28].So study on the pH and its effect on
sperm density and fertilization process is very important to
evaluate its potentials. The assessment of fertilization [30] and
hatching rates [24] can be of valuable indices for reproduction

Percentage of fertilization = (total number of eggs / number of

healthy fertilized eggs) 100
After hatching within 5-6 days according to water
temperature, the larval hatching rate was calculated using
below formula [21]:
Hatching rate = (number of healthy fertilized eggs / number of
fertilized eggs) 100

Sperm density was measured by haemosytometer counting

method was used [8] with a dilution ratio of 1:300 and
observed at 40 X objective magnification. pH was measured
by a table pH meter with the precision of 0.01 pH. All the
experiments in this study were evaluated from three replicates
done at room temperature (22-25 C) and mean values for
each sample referred as indices. To avoid experimental error

H.Dadras : Islamic Azad University Lahijan Branch, Young researcher

club, Lahijan, Iran .P.O.Box:1616
H.Khara, E. shahkar: Islamic Azad University, Lahijan Branch, Fishery
and Aquaculture Department, Lahijan, Iran. P.O.Box :1616.
s. Baradaran Noveiri: International Sturgeon Research Institute.Rasht, Iran.
P.O .Box: 41635-3464.

358

International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2011)

all measurements were randomly done by the same observer

during investigation. Correlation assessments between
parameters were used in the Pearson correlation test
confidence at 5% levels of used by SPSS (Ver. 14) software.
Figures illustrated by Excel (Ver. 2007) software.

10
9
8

Sperm pH

III. RESULTS
Range of sperm density, sperm pH, fertilization and
hatching rates measured in this study were 0.92- 4.4 109
sperm per ml, 7.5 9.09, % 41 95 and % 32 82
respectively. The mean and SD examined parameters are
shown in Table 1. Correlation test between sperm density and
fertilization rate showed a significant negative correlation (r =
0.064, P > 0.05) (Figure 1). Relationship between sperm
density and hatching rate (Figure 2) was also strongly
negative (r = 0.745, P> 0.01). The analysis of data revealed
strong correlations between sperm pH and sperm fertilization
rate (Figure 3) (P <0.05, r = 0.633) and between sperm pH
and hatching rate (P<0.05, r = 0.711) (Figure 4). In all
experiment, analysis of variance showed no significant
differences (P <0.05) between fertilization and hatching rates.

6
5
4

y = 0.0169x + 7.0505
R2 = 0.4011

3
2
1

1T

20

30

40

50

60

70

80

90

100

0T1

0T1

Figure 3: Correlation and regression relationship between fertilization

rate(%) and sperm pHin Acipenser persicus

0T1

0T1

0T1

5T

0T5

0T1

0T1

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T1

10
9
8

0T1

Sperm pH

0T1

Fertilization rate

6
y = 0.0184x + 7.1836
R2 = 0.5049

5
4
3
2
1
0
20

30

40

50

Table 1: MEAN AND STANDARD DEVIATION OF PARAMETERS

OF PERSIAN STURGEON ACIPENSER PERSICUS MILT
SD

2.1 109
8.41

Sperm density (cells/ml)

Sperm pH
Fertilization rate (%)
Hatching rate (%)

1.3 109
0.53

79.1
62.5

100
90

Fertilization rate

80
70
60
50
40
30
y = -9.9268x + 93.414
R2 = 0.4086

10
0
0

Sperm density

Figure 1: Correlation and regression relationship between sperm density (

109 spz/ml) and fertilization rate(%)in Acipenser persicus
5T

0T5

0T5

1T5

0T1

0T5

0T5

0T

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

5T

0T5

90
80
70
60
50
40
30
20
10
0

Hatching rate

0T5

y = -11.972x + 84.662
R 2 = 0.5547

Sperm density

Figure 2: Correlation and regression relationship between sperm density (

109 spz/ml) and hatching rate(%)in Acipenser persicus
5T

0T5

0T1

0T1

0T5

0T5

0T5

0T

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

90

0T5

0T1

0T1

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

0T5

IV. DISCUSSION
Assessment of sperm quality for enhancing the efficiency of
fish artificial reproduction and need for gamets with good
quality to ensure high survival rate of embryos is well
reviewed [5]. Seminal plasma has a unique biochemical
composition to support normal spermatozoan functions [14].
The effect of pH on sperm motility in different fish species
were fully considered by Alavi and Cosson (2005). The
average pH of Persian sturgeon semen in this study was
determined as 8.41 0.53. The individual variations of semen
ionic parameters [4] and sperm density among Persian
sturgeon male fishes have already been reported [8].
According to Gallis et al (1991) semen of Siberian sturgeon
(Acipenser baeri) has an average pH at 8.1 with a high
buffering capacity in this fish. The pH value in white sturgeon
fish (Acipenser transmontanus) also has been reported equal
to 8 [23]. These variations on pH values could be subjected to
semen sampling by abdominal pressure and mixing by urine
[16].
The effect of higher pH on increasing of sperm motility has
been reported on rainbow trout [27], African catfish (Clarias
gariepinus ) [29], Caspian Sea Kutum fish (Rutilus frisii
kutum) [37], Bream (Abramis brama) [40] and goldfish
(Carassius auratus gibelio) [41]. Reduce of pH from 8 to 7.5
in the sperm of Atlantic cod (Gadus morhua), made severe
reduction in percentage of spermatozoa motility and velocity
[17]. Ingermann et al (2002) reported that the initiation of
sperm motility has been inhibited at pH 7.5 and the maximum
motility rate shown at pH 8.2 in white sturgeon

19.93
21.07

20

80

Figure 4: Correlation and regression relationship between hatching rate(%) an

d sperm pHin Acipenser persicus
0T5

Mean

70

Hatching rate

5T

Parameter

60

5T

0T5

359

International Conference on Chemical, Environmental and Biological Sciences (ICCEBS'2011)

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buffering capacity in this fish represents a physiological
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pH (from7.5 to 9/09) and fertilization (from 0.41 to 0.95) and
hatching rates (from 0.32 to 0.82) (Figures 3, 4).
Sperm density is one of the main parameters determining
the success of fertilization and can be a key factor for
assessing the fertilization rates [26]. Our data have shown a
significant decrease of hatching rate by increasing sperm
density (Fig.1), meanwhile decreasing by semen pH
(Fig.4).There has been noted that increasing of sperm density
is highly correlated with fertilization and hatching rates in
many fish species such as Atlantic salmon [22,1], rainbow
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The number of micropiles in Persian sturgeon reported to 89 [20]. So because of polyspermy phenomenon in sturgeons,
there must be a correct sperm to egg ratio to obtain the best
results of fertilization [19,7]. The spermatozoa concentrations
obtained in this study (Tab.1) are close to the cited literature
before [4,8]. Meanwhile studies on relationship between
sperm density and fertilization rate in Atlantic halibut had not
shown any correlations [38].
It is believed that the fertilization results could be affected
by age, weight, size, nutrition and gamete qualities among
different broodstocks [12,14] This study proposed seminal
fluid pH evaluation before breeding could be a valuable
indicator to predict the Persian sturgeon male fertilization
potentials
ACKNOWLEDGMENT
The authors would like to thank Mr. Hossein Mohammadi
for excellent technical assistance of fish sperm sampling at
Shahid Beheshti sturgeon breeding complex (Rasht). We also
gratefully acknowledge the head and staff of Dr. Dadman
International Sturgeon Research Institute (Rasht) for their
appreciated contributions.
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