Fish Physiol Biochem (2009) 35:591–597 DOI 10.1007/s10695-008-9285-3
Structural abnormalities of common carp Cyprinus carpio spermatozoa Martin Psˇenicˇka Æ Marek Rodina Æ Martin Flajsˇhans Æ Vojtech Kasˇpar Æ Otomar Linhart
Received: 17 June 2008 / Accepted: 21 October 2008 / Published online: 9 November 2008 Ó Springer Science+Business Media B.V. 2008
Abstract Spermatozoa of common carp Cyprinus carpio are typically consist of a primitive head without acrosome, a midpiece with several mitochondria, a centriolar complex (proximal and distal centriole), and one flagellum. During an evaluation of the motility of common carp spermatozoa, we found spermatozoa with more than one flagellum and/or ‘‘double head’’ in three different individuals. This may be related to abnormal spermatogenesis. Ultrastructure and physiological parameters of spermatozoa were examined using light microscopy (dark field with stroboscopic illumination), transmission and scanning electron microscopy, and flow cytometry. The recorded pictures and videos were evaluated using Olympus MicroImage software. All spermatozoa with more than one flagellum had a larger head and shorter flagella. They occasionally demonstrated several cytoplasmic channels separating the flagella from the midpiece. Each flagellum was based upon its own centriolar complex, with the connection of the flagellum to the head always at a constant angle. The flagella always consisted of nine peripheral pairs and one central doublet of microtubules. Sperm exhibited
M. Psˇenicˇka (&) M. Rodina M. Flajsˇhans V. Kasˇpar O. Linhart Department of Fish Genetics and Breeding, Research Institute of Fish Culture and Hydrobiology, University of South Bohemia, 38925 Vodnany, Czech Republic e-mail:
[email protected]
a relative DNA content similar to that found in sperm from normal males, with higher coefficients of variation. Although similar abnormalities have been found in livestock, where they were described as a defect in spermiogenesis, no comparable results have been reported in fish. The frequency at which these abnormalities occurs, the fertilization ability of males with defects in spermiogenesis, the influence of these abnormalities on progeny in terms of ploidy level, and the occurrence of deformities warrant further investigation. Keywords Biflagellate/triflagellate spermatozoa Carp spermatozoon Flagellum Polyflagellism Spermiogenesis Sperm abnormality/malformation
Introduction Common carp (Cyprinus carpio L.) males annually produce high amounts of sperm—1.9 ± 0.2 9 1012 spermatozoa per kilogram male body weight. Approximately 95% of this can be collected as the result of regular hormonal stimulation of spermiation (see review by Yaron 1995) during a 9-month period following the completion of spermatogenesis in October (Saad and Billard 1987). Common carp spermatozoa are one of the most primitive among teleost fish (Billard 1986). During spermiogenesis, which is of a very short duration in
123
592
carp, the morphological changes that occur in the spermatids are limited. The carp spermatozoon is characterized by a slightly elongated head without any acrosome, which ultimately becomes elliptical. Because the basic task of the spermatozoan head is to transfer genetic material localized in the nucleoplasma to the egg, an optimal shape and size is a prerequisite for good penetration of spermatozoon throughout the micropyle (Ginsburg 1968). As in other teleost fish, carp spermatozoa do not have any acrosome. The final size of the nucleus is 2–2.5 9 3 lm (Billard 1970). In teleost spermatozoa, it is possible to identify an electrodense nuclear vesicle, caused by incomplete dehydration of the nucleus during spermiogenesis, by electron microscopy. This feature can be species specific and also nutrition dependent, such as has been found in tench Tinca tinca (Psenicka et al. 2006) and barb Barbus barbus (Alavi et al. 2008). The midpiece of the spermatozoon is firmly connected to the head and contains centriolar and mitochondrial segments. In carp, the midpiece is small and surrounds the proximal part of the flagellum. According to Baccetti et al. (1984) and Emel’yanova and Makeeva (1985), it includes cytoplasmic remnants and a rather stable number (seven to nine) of mitochondria. Two centrioles (proximal and distal) have been found to be positioned at constant angles relative to each other: 125° in goldfish Carasius auratus L. (Baccetti et al. 1984),and 140° in tench (Psenicka et al. 2006). The flagellum, 43 lm in length, is separated from the midpiece by a cytoplasmic channel (Lahnsteiner et al. 2003) and is composed of two central and nine peripheral doublet microtubules, the so-called ‘‘9 ? 2 complex’’ (Billard 1970). Some peculiarities in the flagellum have been reported for some species: cells with two flagella have been found in plainfin midshipman Porichtis notatus (Girard) (Stanley 1969) and channel catfish Ictalurus punctatus (Jaspers et al. 1976). The spermatozoa of Cetopsidae, Aspredinidae, and Nematogenyidae are biflagellate, with the flagella located medial to the nucleus. The same structure is found in Amblycipitidae (Lee and Kim 1999), Ariidae, Malapteruridae (Mattei 1991), and Ictaluridae (Poirier and Nicholson 1982; Emel’yanova and Makeyeva 1991a, b). Mansour and Lahnsteiner (2003) described the occurrence of biflagellate spermatozoa in Aspredinidae and Bagridae. However, Lee (1998) and Kim and Lee (2000)
123
Fish Physiol Biochem (2009) 35:591–597
describe the spermatozoa of Bagridaeas as being uniflagellate. The occurrence of biflagellate spermatozoa in Cetopsidae, Aspredinidae, and Nematogenyidae has been described by Spadella et al. (2006). Proximal and distal centrioles are present in all known biflagellate spermatozoa (Spadella et al. 2006). In teleosts, the flagellum is usually single (Jamieson 1991; Mattei 1991). Another abnormal structure in fish is the absence of central microtubules of the flagellum in Anguilliformes and Elopiformes (‘‘9 ? 0’’ structure) (Mattei and Mattei 1975). In Poeciliidae, Janysiidae, Patolontidae, and Embiotocidae (Billard 1970; Stanley 1969; Van Deurs 1975; Lahnsteiner et al. 1997), the plasma membrane often forms one or two lateral fins or ridges, preferentially oriented along the horizontal plane of the flagella defined by the central microtubules (Billard 1970). Such a modification in the flagellum could improve the efficiency of the flagellar propulsion, as discussed by Afzelius (1978). Although spermatozoa of cyprinid fish do not possess a fin, their flagella usually contain vesicles on the base, as found in tench (Psenicka et al. 2006). Massanyi (1991) described abnormalities in the morphology of livestock spermatozoa, but information on fish is lacking. We report here the first results on abnormalities in the structure and motility of carp spermatozoa.
Materials and methods This study was conducted at the Department of Fish Genetics and Breeding, Research Institute of Fish Culture and Hydrobiology (RIFCH), University of South Bohemia, Vodnany, Czech Republic. Semen samples were collected from broodfish reared in the Institute’s fish hatchery. Electron microscopic studies were performed in the Laboratory of Electron Microscopy, Institute of Parasitology, Academy of Sciences of the Czech Republic in Ceske Budejovice. Broodfish and gamete collection Male common carp (Cyprinus carpio) aged 4–7 years old weighing 4–10 kg were collected from a pond and transferred to a 4000-l broodfish tank with recirculating water (0.2 l s-1, 22°C) inside the
Fish Physiol Biochem (2009) 35:591–597
hatchery. Spermiation was induced by a single intramuscular injection of homogenized carp pituitary extract at a dose of 3 mg kg-1 body weight (BW). Sperm were collected 24 h after hormonal injection, transferred to a separate cell culture container (250 ml), and stored at 4°C until observation by means of light microscopy. Abnormal sperm of three males were identified during spawning season and fixed for a later study. Electron microscopy Heterosperm from evaluated males were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 2 days at 4°C, following which it was postfixed, washed repeatedly for 2 h at 4°C in 4% osmium tetroxide, and dehydrated through an acetone series. Samples for scanning electron microscope (SEM) were dehydrated in a critical point dryer pelco CPD 2 (Ted Pella, Redding, CA). Samples were coated with gold under vacuum with an SEM Coating Unit E5100 (Polaron Equipment, Watford, Hertfordshire, UK) and evaluated for morphological parameters using a JSM 6300 scanning electron microscope (JEOL, Akishima, Tokyo, Japan). A JSM 7401 S (JEOL) in cryo-regime was used to identify the parameters in more detail. Samples for transmission electron microscopy (TEM) were embedded in resin (Polybed 812; Polysciences, Warrington, PA). A series of ultra-thin sections were cut using a Leica UCT ultramicrotome (Leica Microsysteme, Austria), double-stained with uranyl acetate and lead citrate, and viewed in a JEOL 1010 TEM (JEOL, Tokyo, Japan) operated at 80 kV. Micrographs were processed using Olympus MicroImage software (ver. 4.0.1; Windows), and the morphological parameters of the spermatozoa were measured.
593
was used as a standard for the relative DNA content of a haploid (1n). Sperm motility Sperm velocity (lm s-1) and percentage of motile sperm after activation (%) were measured using darkfield microscopy (magnification: 2009). The sperm were diluted in distilled water to measure sperm motility. To prevent the sperm from sticking to the slide, we added 0.1% bovine serum albumin (BSA) to the water. Sperm motility was analyzed from a video recording made after activation using a 3 CCD video camera (SONY DXC-970MD, Japan) mounted on a dark-field microscope (NIKON Optiphot 2, Japan). The successive positions of the recorded sperm heads were measured from video frames using a video-recorder (SONY SVHS, SVO-9500 MDP) and analyzed, from five successive frames each, with a micro-image analyzer (Olympus Micro Image 4.0.1. for Windows, Japan). Data analysis Sperm motility and the number of abnormal spermatozoa were measured in triplicate for each sample, and at least 200 spermatozoa from each male were evaluated. An extremely low frequency of abnormal spermatozoa eliminated the analysis of motility parameters of sperm from two of the three males. The data were processed using t-tests for DNA contents and an analysis of variance (ANOVA) for morphological parameters in Statistica ver. 8 (StatSoft, Tulsa, OK). Significance was set at P\0.05.
Flow cytometry
Results
The ploidy level of spermatozoa was determined as relative DNA content using a Partec CCA I flow cytometer (Partec, Mu¨nster, Germany). Fresh sperm were collected into Kurokura immobilizing solution (128.34 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl22H2O, 2.38 mM NaHCO3; Rodina et al. 2004) at a 1:200 ratio. Samples were prepared according to Linhart et al. (2006), and nuclear DNA was stained with 40 ,6-diamidino-2-phenylindole (DAPI). Morphologically normal sperm of common carp (16 males)
Three individuals with spermatozoa containing one to three flagella and/or double-head were identified. The percentages of occurrence of malformed spermatozoa are presented in Table 1. These sperm also showed some differences in ultrastructure, motility, and relative DNA content compared to normal sperm. The spermatozoa ultrastructure of carp with a presumed defect in spermiogenesis could be differentiated into a head or ‘‘double head’’, a midpiece, and one, two, or three flagella. Structures of the head,
123
594
Fish Physiol Biochem (2009) 35:591–597
Table 1 Percentage of abnormal spermatozoa identified in the common carp males evaluated Defect
Male 1 (%)
Male 2 (%)
Male 3 (%)
Biflagellar spermatozoa
9.53
5.14
2.62
Triflagellar spermatozoa
5.30
6.27
1.41
Double-headed spermatozoa
2.35
3.95
0.93
midpiece and part of flagella are shown in Fig. 1a–c. The roundish head of multi-flagellated spermatozoa was clearly larger than that in uni-flagellated ones, but the length of the flagella of the former was generally shorter than than of the uni-flagellated spermatozoa (Table 2). When two or more flagella were present, each flagellum was based on its own centriolar complex composed of proximal and distal centrioles. Cytoplasmic channels were identified around the flagella, formed by an invagination at the plasmalemma that created an extracellular space between the cytoplasmic sheath and the flagellum. Although flagella frequently possessed a separate cytoplasmatic channel, communal channels were identified (Fig. 1b). Some ‘‘double-headed’’ spermatozoa, with their nuclei surrounded by one cytoplasmic membrane, were found (Fig. 2a, b). These spermatozoa contained one or more (usually two) flagella with a communal midpiece, but separate cytoplasmic channels (Fig. 2b). In all malformations, the connection of the flagellum to the head formed a constant angle. Each flagellum had a typical structure of one central pair of singlets and nine peripheral doublets of microtubules. Vesicles occurred around the base of the flagellum (Figs. 1b, 2b). The nucleus of abnormal spermatozoa contained nuclear vesicles, but the number of these tended to fall in the high range of that seen in normal spermatozoa (Figs. 1b, 1c; 2b). However, the average area of mitochondria in polyflagellar spermatozoa was significantly larger (0.52 ± 0.11 lm) than that found in normal spermatozoa (0.41 ± 0.08 lm; P \ 0.01). With the exception of the start of movement, the velocity of polyflagellar spermatozoa was higher than that of normal spermatozoa with one flagellum, and the duration of motility was almost doubled (Fig. 3). The mean relative DNA content in sperm from 16 common carp with morphologically normal spermatozoa was 49.80 ± 1.10, with a coefficient of variation (CV) of 2.82 ± 0.55%, which is the haploid (1n)
123
Fig. 1 Scanning (a) (bar: 5 lm) and transmission (b, c) (bar: 1 lm) electron micrographs showing the ultrastructure of multi-flagellated spermatozoa. White arrows Nuclear vesicles, N nucleus, H single head, MP midpiece, F flagellum, CC cytoplasmic channel, V flagellar vesicle, C centriole
standard. The mean relative DNA content (51.36 ± 0.35; 1.03n) in sperm from males exhibiting presence of abnormal spematozoa was similar (P [ 0.05), with a slightly higher CV (3.84 ± 0.46%).
11
Double-headed spermatozoa
1.99 ± 0.07 b
2.99 ± 0.14 d 3.56 ± 0.74 d
2.72 ± 0.19 c
2.32 ± 0.34 b
1.79 ± 0.07 a
Width of head (lm ± SD)
1.83 ± 0.31 b 0.62 ± 0.14 a
1.82 ± 0.46 c 1.17 ± 0.36 a
a
Number of measurements
0.94 ± 0.15 b
0.88 ± 0.22 b
0.78 ± 0.26 a,b
0.70 ± 0.24 a
0.57 ± 0.18 a 0.95 ± 0.43 b
1.00 ± 0.18 a
Length of midpiece (lm ± SD)
Width of posterior midpiece (lm ± SD)
1.42 ± 0.28 b
Width of anterior midpiece (lm ± SD)
All values are given as the mean ± SD. Values within a column followed by different letters are significantly different at P \ 0.05
16
Triflagellar spermatozoa
2.26 ± 0.33 c
1.75 ± 0.08 a
20 20
Normal spermatozoa
Length of head (lm ± SD)
na
Biflagellar spermatozoa
Kind of defect
Table 2 Morphometric characteristics of the spermatozoa of evaluated males
32.08 ± 2.57 b
24.75 ± 7.21 a
22.35 ± 9.89 a
30.69 ± 2.38 b
Length of flagellum (lm ± SD)
Fish Physiol Biochem (2009) 35:591–597 595
Fig. 2 Scanning (a) (bar: 10 lm) and transmission (b) (bar: 1 lm) electron micrographs showing the ultrastructure of spermatozoa with a ‘‘double head’’ (DH). White arrow Nuclear vesicles, M mitochondria; for other abbreviations, see caption to Fig. 1
Fig. 3 Graph comparing the velocity and duration of motility between uniflagellar and polyflagellar spermatozoa
Discussion
Similar to comparable investigations carried out within the framework of livestock breeding programs,
123
596
we have assessed the quality of sperm in male carp during the selection of males for reproduction. To this end, we looked at both motility characteristics and ultrastructure of the sperm cells. A lack of knowledge in this area could increase defective spermiogenesis in progeny, assuming that such defects are genetically fixed. However, a number of ultrastructural changes, such as the number of nucleus vesicles in the barb, are known to be caused by environmental variables and diet (Alavi et al. 2008). Massanyi (1991) describes several types of genetic malformations in the spermatozoa of livestock. The most prominent, malformations of the acrosome (granular, extensive, small, slack, deformed), are clearly not relevant in a study of carp spermatozoa due to its absence , and the possibility of the occurrence of these malformations in fish spermatozoa that do contain an acrosome, such as sturgeon (Psenicka et al. 2007, 2008), is unknown. A second type of malformation is related to the form and size of the head, which deserves attention in those fish species whose spermatozoa must pass through a micropyle in the egg. The micropyle is only slightly larger than the width of spermatozoon head in most fish species, such as sturgeon (Dettlaff et al. 1993); consequently, those spermatozoa with a bigger head would not pass through the micropyllar channel. However, according to Kudo (1980), the inner aperture of the micropyle (5 lm) in carp theoretically allows the entry of two normal spermatozoa as well as an abnormal spermatozoon. A third possible malformation is related to the midpiece (size and form, eccentric, paraxial, retroaxial, broken), which has important functions in connecting the head to the flagellum (centriolar complex) and providing energy for sperm movement of the spermatozoon (mitochondria). It has an influence on movement and structure of the flagellum. Nevertheless, the midpiece of mammalian spermatozoon has numerous differences compared to that of fish, with the primary one being the number and localization of the mitochondria. Whereas mammalian spermatozoa have 50–70 spirally shaped mitochondria clinging to the flagellum, only two to ten mitochondria are found dispersed in the midpiece of fish spermatozoa, and these are not in physical contact with the flagellum (Baccetti et al. 1984). Due to its more complex structure, the midpiece of mammalian spermatozoa is liable to suffer from more malformations than that found in fish. Nevertheless, we report here a high number of
123
Fish Physiol Biochem (2009) 35:591–597
cytoplasmic channels and centrioles caused by the presence of a high number of flagella in the carp spermatozoa. This last type of malformation involves teratomes, and we report here the presence of teratomes in multi-flagellated and/or double-headed spermatozoa. This maximum frequency of this type of malformation in the spermatozoa of livestock used for breeding is 5% (Massanyi 1991). The presence of multi-flagellated (but not double-headed) spermatozoa is normal in many fish species, such as plainfin midshipman (Stanley 1969) and channel catfish (Jaspers et al. 1976). However, the spermatozoa of carp have always been described as being uniflagellated (Baccetti et al. 1984; Emel’yanova and Makeeva 1985). We have also obtained evidence that polyflagellar spermatozoa have a higher velocity and longer motility time than uniflagellar spermatozoa, possibly due to the higher number of mitochondria in the former. As both polyflagellar and double-headed spermatozoa have a larger head size, we expected that these cells would contain more DNA than their uniflagellar counterparts. However, only the variability was higher. Since the flow cytometer was not equipped with any cell sorting device, by far the majority of cells in the whole sperm samples, even those in which a few abnormal spermatozoa were present, were, in fact, normal sperm cells (82.82, 84.64 and 94.94% for male 1, 2, and 3, respectively). The relative DNA content of these abnormal cells thus appeared as a single histogram peak with a somewhat wider CV. On the basis of our results, abnormal sperm can be distinguished by means of dark-field microscopy and electron microscopy; the higher CV in relative DNA content of abnormal sperm can probably only be used as an auxiliary indicator. Future research should focus on the fertilization ability of males with sperm abnormalities and its impact on progeny. Acknowledgements The study was supported financially by USB RIFCH no. MSM6007665809 and GACR no. 524/06/ 0817
References Afzelius BA (1978) Fine structure of the garfish spermatozoan. J Ultrastruct Res 64:309–314. doi:10.1016/S0022-5320 (78)90039-4
Fish Physiol Biochem (2009) 35:591–597 Alavi SMH, Psenicka M, Rodina M, Policar T, Linhart O (2008) Changes of sperm morphology, volume, density and motility and seminal plasma composition in Barbus barbus (Teleostei: Cyprinidae) during the reproductive season. Aquat Living Resour 21:75–80. doi:10.1051/alr: 2008011 Baccetti B, Burrini AG, Callaini G, Gibertini G, Mazzini M, Zerunian S (1984) Fish germinal cell. I. Comparative spermatology of seven cyprinid species. Gamete Res 10:373–396. doi:10.1002/mrd.1120100405 Billard R (1970) Ultrastructure compare´e de spermatozoides de quelquest poissons te´le´oste´ens. In: Baccetti B (ed) Spermatologia comparata. Quaderno 137:71–80 Billard R (1986) Biology of the gametes of some teleost species. Fish Physiol Biochem 2:115–120. doi:10.1007/ BF02264079 Dettlaff TA, Ginsburg AS, Schmalhausen OI (1993) Sturgeon fishes. Developmental biology and aquaculture. Springer, Berlin Heidelberg, New York Emel’yanova NG, Makeeva AP (1985) Ultrastructure of spermatozoids of some cyprinid fishes (Cyprinidae). J Ichthyol 25(4):137–144 Emel’yanova NG, Makeyeva AP (1991a) Ultrastructure of spermatozoids of some representative catfishes. Vopr Ikhtiol 31:1014–1019 Emel’yanova NG, Makeyeva AP (1991b) Morphology of the gametes of the channel catfish Ictalurus punctatus. Vopr Ikhtiol 31:143–148 Ginsburg AS (1968) Fertilization in fishes and the problem of polyspermy. Moskova, Izdatelnaya Nauka (in Russian) Jamieson BGM (1991) Fish evolution and systematics: evidence from spermatozoa. Cambridge University Press, Cambridge, pp 230–295 Jaspers EJ, Avault JW, Roussel JD (1976) Spermatozoal morphology and ultrastructure of channel catfish, Ictalurus punctatus. Trans Am Fish Soc 150:475–480. doi: 10.1577/1548-8659(1976)105\475:SMAUOC[2.0.CO;2 Kim KH, Lee YH (2000) The ultrastructure of spermatozoa of ussurian bullhead, Leiocassis ussuriensis (Teleostei, Siluriformes, Bagridae). Kor J Limnol 33(4):405–412 Kudo S (1980) Sperm penetration and the formation of a fertilization cone in the common carp egg. Dev Growth Differ 22:403–414 Lahnsteiner F, Berger B, Weismann T, Patzner R (1997) Sperm structure and motility of the freshwater teleost Cottus gobio. J Fish Biol 50:564–574 Lahnsteiner F, Berger B, Weismann T (2003) Effects of media, fertilization technique, extender, straw volume, and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen. Theriogenology 60:829–841. doi: 10.1016/S0093-691X(02)01300-6 Lee YH (1998) Ultrastructure of spermatozoa in the bagrid catfish, Pseudobragrus fulvidraco (Teleostei, Siluriformes, Bagridae). Korean J Electron Microsc 28(1):39–48 Lee YH, Kim KH (1999) Ultrastructure of the south torrent catfish, Liobagrus mediadiposalis (Teleostei, Siluriformes, Amblycipitidae) spermatozoon. Kor J Limnol 32: 271–280
597 Linhart O, Rodina M, Flajshans M, Mavrodiev N, Nebesarova J, Gela D, Kocour M (2006) Studies on sperm of diploid and triploid tench (Tinca tinca L.). Aquacult Int 14:9–25. doi:10.1007/s10499-005-9010-5 Mansour N, Lahnsteiner F (2003) Morphology of the male genitalia and sperm fine structure in siluroid fish. J Submicrosc Cytol Pathol 35(3):277–285 Massanyi L (1991) Funkcˇna´ morfolo´gia spermie. VEDA-Vydavatelstvo Slovenskej Akademie Vied, Bratislava Mattei X (1991) Spermatozoon ultrastructure and its systematic implications in fishes. Can J Zool 69(12):3038–3055. doi: 10.1139/z91-428 Mattei C, Mattei X (1975) Spermiogenesis and spermatozoa of the Eiopomorpha (teleost fish). In: Afzelius BA (ed) The functional anatomy of the spermatozoon. Oxford, Pergamon Press, pp 211–221 Poirier GR, Nicholson N (1982) Fine structure of the testicular spermatozoa from the channel catfish, Ictalurus punctatus. J Ultrastruct Res 80:104–110. doi:10.1016/S0022-5320 (82)80036-1 Psˇenicˇka M, Rodina M, Nebesarova J, Linhart O (2006) Ultrastructure of spermatozoa of tench Tinca tinca observation with scanning and transmission electron microscopy. Theriogenology 64:1355–1363. doi:10.1016/ j.theriogenology.2006.04.040 Psˇenicˇka M, Alavi SMH, Rodina M, Gela D, Nebesarova J, Linhart O (2007) Morphology and ultrastructure of Siberian sturgeon, Acipenser baerii, spermatozoa using scanning and transmission electron microscopy. Biol Cell 99:103–115. doi:10.1042/BC20060060 Psˇenicˇka M, Vancova M, Koubek P, Linhart O (2008) Fine structure and morphology of sterlet (Acipenser ruthenus L. 1758) spermatozoa and acrosin localization. Anim Reprod Sci. http://dx.doi.org/10.1016/j.anireprosci.2008.02.006 Rodina M, Cosson J, Gela D, Linhart O (2004) Kurokura solution as immobilizing medium for spermatozoa of tench (Tinca tinca L.). Aquacult Int 12:119–131. doi: 10.1023/B:AQUI.0000017192.75993.e3 Saad A, Billard R (1987) The composition and use of a sperm diluent in the carp, Cyprinus carpio. Aquaculture 66: 329–345. doi:10.1016/0044-8486(87)90117-7 Spadella MA, Oliveira C, Quagio-Grassiotto I (2006) Occurrence of biflagellate spermatozoa in the Siluriformes families Cetopsidae, Aspredinidae, and Nematogenyidae (Teleostei: Ostariophysi). Zoomorphology 125:108–118. doi:10.1007/s00435-006-0018-9 Stanley HP (1969) An electron microscope study of spermiogenesis in the teleost fish Oligocottus moculosus. J Ultrastruct Res 27:230–243. doi:10.1016/S0022-5320 (69)80014-6 Van Deurs B (1975) The sperm cell of Pantodon (Teleostei) with a note residual body formation. In: Afzelius BA (ed) The functional anatomy of the spermatozoon. Pergamon Press, Oxford, pp 311–318 Yaron Z (1995) Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129:49–73. doi:10.1016/0044-8486(94)00229-H
123
