Spermatozoa characterization in the one-sided livebearing Jenynsia multidentata (Cyprinodontiformes: Anablepidae)
Caracterización de parámetros espermáticos del pez vivíparo Jenynsia multidentata (Cyprinodontiformes: Anablepidae)
María A. Roggio1*, María E. Teves2*, Andrea C. Hued1, Laura C. Giojalas3* & ]]>
1
Abstract
Several sperm parameters have been ]]>
Jenynsia multidentata (Anablepidae) inhabits an extensive area of the Neotropical region and it has been used as a useful fish laboratory model to evaluate the effects of xenobiotics through different biomarkers. The present work characterized the sperm ]]>
J. multidentata populations.
Diversos parámetros espermáticos han sido utilizados para evaluar la fertilidad de peces. Dentro de los peces ]]>
Jenynsia multidentata (Anablepidae) habita una extensa área de la región Neotropical y ha sido utilizado como un exitoso modelo ]]>
J. multidentata. ]]>
Palabras clave: parámetros espermáticos, morfometría, motilidad espermática, peces vivíparos, Jenynsia multidentata.
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Spermatozoa structure in teleost species reveals a high diversity, mainly at the family level. The spermatozoa of internal and external fertilizers differ in their organization. External fertilizers have a simpler organization, show- ing an ovoid or spherical nucleus, and a small midpiece containing only few mitochondria, whereas species with internal fertilization have an elongated nucleus and a relatively bigger midpiece (Lahnsteiner & Patzner, 2008; Jamie- son, 1991, 2009).
Within teleosts, approximately 3% of fish species are known to be viviparous (Wourms, 2005). Particularly, the order Cyprinodontiformes includes several species with internal fertilization (Parenti, 2005). Within this order, most of the studies about sperm characteristics have focused on the family Poeciliidae (Grier, 1973, 1975; Constantz, 1984; Meffe & Snelson, ]]>
The one-sided livebearing fish, Jenynsia multidentata (Jenyns 1842) (Anablepidae) has a wide distribution in an extensive area of the Neotropical region (Ghedotti, 1998) inhabiting both polluted and non-polluted areas (Hued & Bistoni, 2005). It presents sexual dimorphism; males are smaller than females and have a modificated anal fin, called gonopodium (Galindo-Villegas & Sosa-Lima, 2002). Mating behavior is coercive; males approach females from behind and try ]]>
J. multidentata
feeds on mosquito larvae (Ringuelet, Aramburu, & de Aramburu, 1967; Marti, Azpelicueta, Tranchida, Pelizza, & Garcia, 2006).
Spermatozoa characterization offers a use- ful tool to evaluate the fertility potential of male fish. It has been demonstrated that sperm quality could be determined by sperm count, motility, ]]>
J. multidentata by dynamic parameters, sperm count, viability and morphometry, through a simple protocol of semen collection and to establish basic parameter values for the evaluation of the reproductive ]]>
J. multidentata populations.
Materials and Methods
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Semen collection: Forty-five adult males of J. multidentata (mean standard length: 28.83±3.41mm; mean body weight: 0.479±0.131g) were captured by a backpack electrofisher from a site on Yuspe River, Córdoba, Argentina (31°14’1”8 S - 64°31’14” W), during a ]]>
Sperm dynamic parameters: Immediately after semen extraction, 12µL aliquot of diluted sperm suspension was placed on a glass slide. The samples were recorded at 100x magnification during four minutes, with a random change of the microscope field every ten seconds. Sperm analysis was carried out with a videomicroscopy system consisting of a phase ]]>
Viability and sperm count: Fifteen ]]>
The volume of semen obtained from each individual, determined by observation of fluid height in the capillary, was approximately the same (about 2µL). A sperm sample dilution of 1:10 was prepared in distilled water and placed in an “improved Neubauer chamber” haemocytometer in ]]>
Sperm morphometry: 20µL of sperm sample were fixed with 2% formaldehyde and then stained with Coomassie-blue (220% wt/ vol). The microphotographs were taken at 1 000x magnification using a light microscope (Olympus® CX31) and a ]]>
In order to corroborate the spermatozoa lengths, gonopodium of five males were fixed with 2% of glutaraldehyde and 4% formalde- hyde in 0.1M cacodylate buffer for 2h, ]]>
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Descriptive statistical measures were obtained through the software package InfoStat (2011). Values are presented as means±standard deviation (SD).
Results
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Forty-five males presented mean values of VSL and VCL of 81.67µm/s (SD=3.53) and 85.73mm/s (SD=3.55), respectively (Fig. 1A, Fig. B). A linear pattern of movement greater than 89% were observed in all sperm samples, showing a linearity between 94 and 98% in 75% of samples. A proportion of live spermatozoa higher than 60% was ]]>
Fig. 1C). The mean percentage of live mobile and immobile spermatozoa were 68.28% (SD=8.32%) and 9.46% (SD=4.56%), respectively. On the other hand, the spermatozoa count showed a high variability between males, being the average value of the 5 524cells/µL sample (SD=728) (Fig. 1D).
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The morphometry analyses showed a total sperm length of 46.66μm (SD=2.06), a head length of 3.46μm (SD=0.41) and a midpiece region of 9.12μm (SD=0.65) (Table 1; Fig. 2A, Fig. 2C). The relative frequency of morphometrical values are shown in figure 3. The 65% of ]]>
Fig. 3A). A midpiece region length values between 9 and 10μm were registered in 50% of males (Fig. 3C).
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Discussion
The present work characterized the spermatozoa of J. multidentata through a simple protocol of semen ]]>
Males of J. multidentata present tubular gonopodium, an enclosed tube that enables sperm transfer during copulation (Turner, 1950; Grier et al., 1981; Malabarba, Reis, Vari, Lucena, & Lucena, 1998). Therefore, spermatozoa are not packaged as spermatozeugmata or spermatophores as occurs in Poeciliidae, but they are released as clumps within mucilaginous material (Grier et al., 1981). Although the sperm ultrastructure has been described by Dadone & Narbaitz (1967), these authors did report neither morphometrical values nor dynamic parameters. ]]>
It is known that the spermatozoa of inseminating fish present some differences when compared with externally fertilizing species, which appear to be correlated with the mode of insemination (Jamieson, 1991, 2009; Burns & Weitzman, 2005). Species with internal fertilization have a more complex sperm orga- nization, an elongated sperm nucleus and a relatively larger midpiece region compared to externally fertilizing fishes (Jamieson, 1991; Mattei, 1991; Lahnsteiner & Patzner, 2008).
Spermatozoa of J. multidentata share a similar morphology with the general model described for inseminating fish. We registered a total length of spermatozoa of around 46.66µm. Comparing with other viviparous species within the same order, the total length is longer ]]>
Anableps anableps Linnaeus, 1758 (40μm) (Greven & Schmahl, 2006) but shorter than in Poecilia reticulata Peters, 1859 (54.56μm) (Skinner & Watt, 2007) and Xiphophorus nigrensis Rosen, 1960 (57.7μm) (Smith & Ryan, 2010). Similar length (around 50µm) has been registered in viviparous fish of another order such as Cymatoguster aggregate (Perciformes, Embiotocidae) (Gardiner, 1978). On ]]>
The head length of J. multidentata ]]>
X. nigrensis which has a shorter head length (2.70µm approximately). Also, this parameter is longer than most of the external fertilization fishes which present a head length lesser than 2µm (Hadi-Alavi et al., 2009; Lahnsteiner & Patzner, 2008). An elongated head, such as the recorded in
J. multidentata, is a common feature shared by most fishes with internal fertilization. This characteristic gives the sperm certain advantages to move into the female reproductive tract, such as the increase of the side-to-side alignment which enables the clumping of the cells allowing spermatozoa to flow together, and the increase of the direc- tionality of cell movement (Ginzburg, 1968; Gardiner, 1978).
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The midpiece of J. multidentata (9.12μm) is similar to that registered in X. nigrensis (8.94μm approximately) (Smith & Ryan, 2010) but is longer than the values recorded for P. reticulata, and A. anableps (4.79 and 3.9μm, ]]>
C. aggregate (approximately 2µm) (Gardiner, 1978) and external fertilization fishes, where the mid- piece is less than 2µm (Lahnsteiner & Patzner, 2008; Hadi-Alavi et al., 2009). In viviparous fish, it has been proposed that an enlarged midpiece increases the capacity of the sperm’s energy-generating mechanism and might help to prolong the life-span of the spermatozoa ]]>
Sperm fertility has been related to sperm motility in several fish species. Sperm motility, evaluated as the sperm velocity and the percentage of motile spermatozoa, is an integrative quality parameter which combines ]]>
The high sperm linearity observed in J. multidentata is similar to other fish species with either external or internal fertilization (Lahnsteiner & Patzner, 2008). These authors proposed that the ]]>
J. multidentata could be an adaptation to sperm competition pressures. Therefore, in internally fertilizing species with female sperm storage, sperm motility would be important in determining paternity because more motile sperm can remain longer in the female tract (Snook, 2005; Evans, Pilastro, & Schlupp, 2011).
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The sperm count recorded in J. multidentata presented a great variation among individuals. Our results were in agreement with the high heterogeneity reported by Rurangwa et al. (2004) in external fertilization fishes such as Oncorhynchus mykiss, Cyprinus carpio and Acipenser ]]>
These authors pointed out that sperm concentration is not a sensitive or specific measure of sperm fertilizing capacity, as the concentration can vary greatly within a fish species and across the reproductive season. Copulation in poeciliids is rapid (<1s) and does not involve male mounting or clasping that may increase male control over sperm transfer (Birkhead & Møller, 1998). In this regard, J. ]]>
also presents the same behavior. These results suggest that female behavior is often effective in limiting the size of the ejaculate transferred by finishing the copulation early. Therefore, it has been proposed that traits that increase sperm quality, such as viability and motility (e.g. spermatozoa velocities), as well as ejaculate size or the number of sperm produced might evolve in species in which males have little control over the amount of sperm inseminated (Pilastro, Gas- parini, Boschetto, & Evans, 2008; Gasparini, ]]>
Several of the parameters discussed above have been used as useful tools to evaluate fish fertility (Billard & Cosson, 1992; Kime & Nash, 1999; Rurangwa et al., 2004). The evaluation of seminal quality constitutes a critical step in species management and conservation. The results of our work have established the basic parameter values to be in use in the evaluation ]]>
J. multidentata. Since this species is widely distributed in both polluted and non-polluted sites and has been used as a bioindicator in water quality assessment, the most of the sperm parameters characterized in the present work could be used as a sensitive set of indirect biomarkers that could provide early warning signal of reproductive alterations in polluted freshwater systems of an extensive area of the ]]>
Acknowledgments
We are grateful to Laura V. Gatica, Héctor A. Guidobaldi and Diego R. Uñates for laboratory ]]>
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1. Cátedra de Diversidad Animal II, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba - Instituto de Diversidad y Ecología Animal, CONICET, Av. Vélez Sarsfield 299, CP X5000JJC, Córdoba, Argentina; angelinaroggio@gmail.com, achued@efn.uncor.edu, mbistoni@efn.uncor.edu 2. Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, 23298, USA; eugeteves@yahoo.com.ar 3. Centro de Biología Celular y Molecular, Edificio de Investigaciones Biológicas y Tecnológicas, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, X5016GCA, Córdoba, Argentina; lcgiojalas@com.uncor.edu ]]>
Received 01-III-2013. Corrected 09-II-2014. Accepted 03-III-2014. ]]>2009741179-118620121992261122-13119982000106961-9782010165535-54820045665-702005105-1322008421299-13071984465-4851999161-18619678214-2192005311-9200378-61220111970290-12720041444-47200250441151-1157197813435-43820105881-51998561-5821968200683-8819734582-92197516589-1021981797-80120097232-43543279-298201262107-117Infostat20111991200981999233123-12920081-611998200721140-46200631102-1061991693038-305519891993254153-1622005105-132199475179-18520081922374-381196720042341-2820071847-522010231759-177120052046-53195086329-3662002211353-366World Health Organization201052005238-26219954258-64