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ISSN 1303-2712
Turkish Journal of Fisheries and Aquatic Sciences 16: 947-951 (2016) DOI: 10.4194/1303-2712-v16_4_22

RESEARCH PAPER

A Preliminary Study on Sperm Morphology, Motility and Composition of

Seminal Plasma of Shirbot, Barbus grypus

Mojgan Khodadadi1,*, Aida Arab1, Amir Jaferian1

1
Islamic Azad University, Department of Fisheries, Ahvaz Branch, Ahvaz, Iran.

* Corresponding Author: Tel.: +989166066295; Received 12 May 2016

E-mail: [email protected] Accepted 19 June 2016

Abstract

In the present study, we investigated some qualitative parameters of milt in an endemic cyprinid fish, Shirbot, Barbus
grypus. The parameters including: sperm motility, sperm density and the composition of seminal plasma (calcium, sodium
and potassium, phosphorous, cholesterol, glucose. In this regard, 13 males ready to spawning (TW: 2846 ± 324 g, TL: 65.8 ±
4.8 cm) were provided from the South Aquaculture Research Center. Then, fish were injected during 12 days intraperitoneally
in one stage with 2 mg/kg.bw pituitary extract and after 10-12 h, the spermiation occurred and sperm collection was carried
out. According to results, the mean values of assayed parameters were: sperm motility duration: 108±13.6 s; sperm density:
18.8±2.44; seminal plasma pH: 7.87±0.2; % spermatocrit: 76.07±9.57; glucose: 9.41±1.29 mg/dl; cholesterol: 25.71±2.99
mg/dl; total protein: 1.27±0.29 mg/dl; Na+: 93.73±7.78 mmol/l; K+: 44.3±4.5 mmol/l; Ca++: 0.86±0.06 ; Mg++: 1.43±0.18
mmol/l; total sperm length: 4.17±0.34 µm; sperm head length: 1.2±0.2 µm; sperm flagellum length: 3.01±0.21 µm. Also,
significant correlation was found between sperm motility duration and pH of seminal plasma and also spermatocrit and sperm
density.

Keywords: Endemic fish, sperm, seminal plasma, Barbus grypus.

Introduction correlation between % sperm motility and Na+, K+, pH,

osmolality in rainbow trout, Onchorhynchus mykiss
(Lahnsteiner et al. 1998), % sperm motility and pH in
The endemic fish species are ecologically important
Chinook salmon, Oncorhynchus tshawytscha (Ingermann et
and they considered as gene banks of an ecosystem.
al., 2002), % motility vs. Na+, K+, total protein, pH and
Knowledge on all biological aspects of these species
osmolality in Alburnus alburnus (Lahnsteiner et al. 1996)
especially reproduction properties may help to their
and also negative correlations between the % motility and
appropriate management and conservation in the nature. In
the concentrations of Ca2+, Mg2+, Na+, K+ in European eel,
this regards, many studies have investigated the
Anguilla anguilla (Perez et al. 2003). A recent study by
reproduction properties of fishes especially gamete quality,
Öğretmen et al. (2014) indicated that motility of shabut,
the studies which followed the aquacutural and
Barbus grypus spermatozoa is in under influence of the
conservational goals .Sperm motility and sperm density
composition of activation solution especially K+ and Ca++
determine the fertilization capability of spermatozoa and are
concentrations. In this regard, the concentrations more than
considered as the qualitative parameters of fish spermatozoa
20 mM K+ and 10 mM Ca++ significantly decreased the
(Suquet et al., 1982; Billard et al., 1993; Linhart et al.,
sperm motility of shabut.
1994; Krol et al., 2006). Seminal plasma has a special
The Shirbot, Barbus grypus is one of the endemic and
composition composed of organic and inorganic (ions)
freshwater fish of Iran. This species is nutritionally
components which support the viability of spermatozoa
omnivorous and ecologically euryhaline and eurytherme
(Piironen and Hyvarinen, 1983; Lahnsteiner et al., 1993
and widely distributed in Iran, Turkey, Syria and Iraq
(a,b); Ciereszko et al., 2000). K+ is a key ion controlling
(Nikpei, 1996; Abdoli, 2000). Shirbot is favorable among
sperm motility in Salmonidae and Acipenseridae in
indigenous residents of south western provinces of Iran,
combination with osmotic pressure (Alavi et al., 2006). In
especially Khuzestan Province (Banaee and Naderi, 2014).
cyprinidae, the sperm motility is under control of seminal
The growth rate of this species is high and sometimes
plasma osmolality and the motility is prevented when the
reaches up to 20 Kg. Thus, Shirbot can be a good candidate
osmotic pressure is high (Alavi et al., 2006). Many studies
for aquaculture in the southern regions of Iran.
have shown that the composition of seminal plasma affects
Understanding the reproduction properties of Shirbot can
the sperm motility of fishes. In this regard, interesting
help to their management and conservation in the nature and
results were found in fish species, for example, the positive
also to artificial reproduction in hatchery condition. Thus, in
© Published by Central Fisheries Research Institute (CFRI) Trabzon, Turkey
in cooperation with Japan International Cooperation Agency (JICA), Japan
948 M. Khodadadi et al. / Turk. J. Fish. Aquat. Sci. 16: 947-951 (2016)
the present study we investigated some reproductive min in a D-78532 centrifuge (Tuttlingen, Zentrifugen,
parameters including sperm motility, sperm density and the Germany) and then spermatocrit was calculated on the
composition of seminal plasma (calcium, sodium and basis of the ratio of spermatozoa volume (white part) to
potassium, phosphorous, cholesterol, glucose). total volume of milt × 100. To assay sperm density, at first
milt samples were diluted 1:1000 times by pipetting10 µl
Materials and Methods semen in 990 µl of water (Ciereszko and Dabrowski, 1993).
Then, a haemocytometer counting chamber was used to
The experiment was carried out at the South determine the spermatozoa density. A droplet of the diluted
Aquaculture Research Center, Ahvaz, Iran during May to milt was placed on a haemocytometer slide (depth 0.1 mm)
April. Altogether, 13 males ready to spawning (TW: 2846 with a coverslip and counted using light microscopy
± 324 g, TL: 65.8 ± 4.8 cm) equipped to monitor according to Alavi et al. (2006). The
were captured from nature (Karun river) and then light microscope equipped to monitor (400 X) with
transported to the South Aquaculture Research Center. After accuracy of 0.01 µm was used to investigate the
3 days acclimation to pond condition, male fish were morphological properties of spermatozoa including total
injected during 12 days intraperitoneally in one stage with 2 length, head length and flagellum length.
mg/kg.bw pituitary extract. Before injection, the fish were The seminal plasma concentrations of calcium and
anaesthetized using 100 pp of MS222 (tricaine methane phosphorous were assayed photometrically using
sulphonate). After injection, males were checked every 6 h SELECTRA XL automated chemistry analyzer (ELITech
interval up to spermiation. During the course of the group, vital scientific, Chicago, IL (USA) according to…
experiment, the water condition (temperature: 24-25 °C, the protein, cholesterol and glucose were measured
dissolved oxygen: 8-8.5 mg/l and pH: 7.2-7.3) of each pond spectrophotometrically (RA 1000 Technicon) using
was checked and was in normal range. After 10-12 h, the standard analysis kits (standard analysis kits from
spermiation occurred and sperm collection was carried out Parsazmoon, Tehran, Iran). Also, the concentration
by hand-stripping. In this regard, the milt collection was of sodium and potassium were assayed by an automatic
carried out by massage from the anterior portion of the belly electrolyte analyzer (Convergys ISE NG, GmbH,
(testis region) towards the genital papilla. Special care was Germany).
taken to avoid the contamination of semen by water, mucus,
blood cells, faeces or urine. Immediately after milt Statistical Analysis
collection, the pH of milt samples was measured with a pH
meter. Then an aliquot of milt samples were considered for The SPSS software was used to analyze data. All
motility and the sperm density analysis and remaining correlations were tested using the bivariate correlation
centrifuged (13000 g for 10 min) to separate seminal coefficients of Pearson. Then, linear and non-linear
plasma. To evaluate the duration of sperm motility, a 100 µl regression models were investigated using regression fits.
freshwater placed on a glass slide under light microscopy
(Nikon eclipse 50i) equipped to monitor and then 10 µl Results
fresh sperm was added using a microsampler. Then, the
duration of sperm motility was recorded by a time recorder According to our results, the range of all assayed
(Secer 2004). Only forward-moving spermatozoa were parameters (n=13) were as follow: sperm density: 15-23 ×
classified as motile, while sperm cells simply vibrating or 109 ; spermatocrit: 60-90 %; duration of sperm motility: 90-
turning on their axes (circular movement) were considered 132 s; milt volume: 2.9-4.5 ml; seminal plasma pH: 7.65-
as immotile (Aas et al., 1991). 8.27; glucose: 8.2-12.2 mg/dl; cholesterol: 21-29.3 mg/dl;
The spermatocrit is defined as the ratio of white total protein: 0.97-1.9 ; Na+: 84.45-103 mM/l; K+: 36.8-49.3
packed material volume to the total volume of semen × 100 mM/l; Ca++: ; Mg++:1.2-1.55 mM/l; total sperm length:
(Rurangwa et al., 2004). Microhaematocrit capillary tubes 3.48-4.66 µm; sperm head length: 0.88-1.33 µm; sperm
(75 mm in length and 1.1–1.2 mm in diameter) were used flagellum length: 12.6-13.3 µm. Also, the mean ± SD all
for spermatocrit measurement. Microhaematocrit capillary parameters have been presented in Table 1. Also, significant
tubes filled with milt were centrifuged at 5000 rpm for 10 correlation was found between sperm motility duration and

Table 1. Milt parameters of Shirbot, Barbus grypus

Milt parameters Mean±SD

sperm density (× 109) 18.8±2.44
Spermatocrit (%) 76.07±9.54
duration of sperm motility (s) 108±13.6
milt volume (ml) 3.95±0.54
seminal plasma pH 7.87±0.2
Glucose (mg/dl) 9.41±1.29
Cholesterol (mg/dl) 25.71±2.99
total protein (mg/dl) 1.27±0.29
Na+ (mmol/l) 93.73±7.78
K+ (mmol/l) 44.3±4.5
Ca++ (mmol/l) 0.86±0.06
Mg++ (mmol/l) 1.43±0.18
total sperm length (µm) 4.17±0.34
sperm head length (µm) 1.2±0.2
sperm flagellum length (µm) 13.01±1.21
 M. Khodadadi et al. / Turk. J. Fish. Aquat. Sci. 16: 947-951 (2016) 949
seminal plasma pH (Figure 1); sperm motility duration and study, some reproductive properties of an endemic fish
glucose concentration (Figure 2) and also spermatocrit and species, Shirbot, Barbus grypus. In our study, the values of
sperm density (Figure 3). K+ and Ca++ were at the rage of those assayed in other
cyprinidae (K+: 20-78.87 mmol/l; Ca++: 0.7-10.6 mmol/l)
Discussion (Alavi and Cosson 2006). However, the Ca++ concentration
was lower than many cyprinid fishes (Alavi and Cosson
2006). The values of Na+ and Mg++ in Shirbot were higher
Knowledge on biological aspects of fish especially
than other cyprinid fish (Alavi and Cosson 2006) (Na+:
reproductive properties is essential to their management and
51.3-71.2 mmol/l; Mg++: 0.02-0.3 mmol/l). The interactions
conservation in the nature. In this regard, attentions to
of ions present in the seminal plasma with the sperm
endemic fish species are very important since these species
membrane affects the membrane potential (Ciereszko et al.,
are of the most important part of local ecosystem. These
2000) and represent a mechanism of inhibition of
species help to sustain the parts of ecosystem. In the present

140
Duration of sperm motility (s)

130

120

110

100 y = 65.556x - 408.28

R² = 0.9928
90

80
7.5 7.6 7.7 7.8 7.9 8 8.1 8.2 8.3 8.4

pH of seminal plasma
Figure 1. The relationships between seminal plasma pH and duration of sperm motility in Shirbot, Barbus grypus.

140

130
Duration of sperm motility (s)

120

110

100 y = 10.373x + 9.7736

R² = 0.922
90

80
7 8 9 10 11 12 13
Glucose concentration (mg/dl)
Figure 2. The relationships between glucose concentration and duration of sperm motility in Shirbot, Barbus grypus.

95
90
85
Spermatocrit (%)

80
75
70
65
y = 3.8938x + 2.6942
60 R² = 0.9873
55
50
13 15 17 19 21 23 25
Sperm density ( * 109 )
Figure 3. The relationships between sperm density and spermatocrit in Shirbot, Barbus grypus.
950 M. Khodadadi et al. / Turk. J. Fish. Aquat. Sci. 16: 947-951 (2016)
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