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Spermatozoa Characterization in the One-Sided Livebearing Jenynsia Multidentata (Cyprinodontiformes: Anablepidae)

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Spermatozoa Characterization in the One-Sided Livebearing Jenynsia Multidentata (Cyprinodontiformes: Anablepidae) Spermatozoa characterization in the one-sided livebearing Jenynsia multidentata (Cyprinodontiformes: Anablepidae) María A. Roggio1, María E. Teves2, Andrea C. Hued1, Laura C. Giojalas3 & María A. Bistoni1* 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; [email protected], [email protected], [email protected] 2. Department of Obstetrics and Gynecology, Virginia Commonwealth University, Richmond, VA, 23298, USA; [email protected] 3. Centro de Biología Celular y Molecular, Edificio de Investigaciones Biológicas y Tecnológicas, Universidad Nacional de Córdoba, Av. Vélez Sarsfield 1611, X5016GCA, Córdoba, Argentina; [email protected] * Correspondence Received 01-III-2013. Corrected 09-II-2014. Accepted 03-III-2014. Abstract: Several sperm parameters have been employed as useful tools to evaluate fish fertility. Within tele- osts, approximately 3% of fish species are known to be viviparous. The Order Cyprinodontiformes includes sev- eral species with internal fertilization, and within this group most of the studies about sperm quality have been mainly focused on the Poeciliidae family. The livebearing fish 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 of this species through a simple protocol of semen collection. Sperm population showed linearity greater than 89% and 70% of fish have a straight line and curvilinear velocity valued between 50 and 100µm/s. Although 85% of individuals showed a proportion of live sperm higher than 60%, the male population had a high degree of heterogene- ity in its sperm count. Morphometry analyses showed a total sperm and head lengths of 46.66±2.06µm and 3.46±0.41mm, respectively. A rather long midpiece region (9.12±0.65µm) was registered, which may indicate high energy-producing capabilities of the spermatozoa. This study established basic parameter values which could be useful for evaluating reproductive potential of J. multidentata populations. Rev. Biol. Trop. 62 (3): 997-1006. Epub 2014 September 01. Key words: sperm parameters, morphometry, sperm motility, sperm viability, viviparous fish, Jenynsia multidentata. Spermatozoa structure in teleost species 2005). Particularly, the order Cyprinodonti- reveals a high diversity, mainly at the family formes includes several species with inter- level. The spermatozoa of internal and external nal fertilization (Parenti, 2005). Within this fertilizers differ in their organization. External order, most of the studies about sperm char- fertilizers have a simpler organization, show- acteristics have focused on the family Poe- ing an ovoid or spherical nucleus, and a small ciliidae (Grier, 1973, 1975; Constantz, 1984; midpiece containing only few mitochondria, Meffe & Snelson, 1989; Meyer & Lydeard, whereas species with internal fertilization have an elongated nucleus and a relatively bigger 1993; Evans, Pilastro, & Ramnarine, 2003), midpiece (Lahnsteiner & Patzner, 2008; Jamie- whereas within the Anablepidae family, the son, 1991, 2009). few existing studies have focused on sperm Within teleosts, approximately 3% of fish ultrastructure only (Dadone & Narbaitz, 1967; species are known to be viviparous (Wourms, Greven & Schmahl, 2006). Thus, spermatozoa Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (3): 997-1006, September 2014 997 morphometry and dynamic parameters within 0.479±0.131g) were captured by a backpack this family are still unknown. electrofisher from a site on Yuspe River, Cór- The one-sided livebearing fish, Jenynsia doba, Argentina (31°14’1”8 S - 64°31’14” multidentata (Jenyns 1842) (Anablepidae) has W), during a reproductive season (September a wide distribution in an extensive area of the to April) (Mai, Garcia, Vieira, & Mai, 2007; Neotropical region (Ghedotti, 1998) inhabiting Bianco, Guyón, & Bistoni, 2011). Fish were both polluted and non-polluted areas (Hued transported to the laboratory in plastic water & Bistoni, 2005). It presents sexual dimor- tanks (20L). Samplings were performed every phism; males are smaller than females and two months. Males were acclimated during two have a modificated anal fin, called gonopodium weeks under controlled laboratory conditions (Galindo-Villegas & Sosa-Lima, 2002). Mating (temperature at 21ºC; light/dark cycle of 12:12 behavior is coercive; males approach females h) and were fed daily with commercial fish from behind and try to thrust their copula- food (TetraMin®). At the end of the acclima- tory organ in the female genital pore (Bisazza, tization period, each male was anaesthetized Manfredi, & Pilastro, 2000). It is important to in a water solution of MS-222 (5g /L) (Tric- note that this species has been used as a useful aine methanesulfonate; Sigma Aldrich). The fish laboratory model to evaluate the effects gonopodium was swung forward and intro- of xenobiotics through different biomarkers duced in a capillary tube. In order to release (Cazenave et al., 2008; Amé, Galanti, Bocco, & sperm, gentle pressure was applied to the side Wunderlin, 2009; Hued, Oberhofer, & Bistoni, of the abdomen using a cotton tip. The sperm 2012). On the other hand, this fish is considered was collected at the base of the gonopodium. a useful species in controlling mosquito popu- The spermatozoa of this species are not pack- lations since J. multidentata feeds on mosquito aged as spermatozeugmata or spermatophores larvae (Ringuelet, Aramburu, & de Aramburu, but they are released as clumps within mucilag- 1967; Marti, Azpelicueta, Tranchida, Pelizza, inous material (Grier, Burns, & Flores, 1981). & Garcia, 2006). This action was repeated five times for each Spermatozoa characterization offers a use- fish to ensure that all available sperm had been ful tool to evaluate the fertility potential of collected. Sperm samples were suspended in male fish. It has been demonstrated that sperm 80µL of HAMF-10 culture medium, at pH 7.4 quality could be determined by sperm count, (Invitrogen, Argentina) and sperm separation motility, viability and morphology (Kime & was achieved by mixing the suspension with Nash, 1999; Burness, Casselman, Schulte- a micropipette. All measurements were carried Hostedde, Moyes, & Montgomerie, 2004; out at room temperature (21±2°C). Gage et al., 2004; Rurangwa, Kime, Ollevier, & Nash, 2004; Snook, 2005). The main goal of Sperm dynamic parameters: Immedi- the present study was to characterize the sper- ately after semen extraction, 12µL aliquot matozoa of J. multidentata by dynamic param- of diluted sperm suspension was placed on eters, sperm count, viability and morphometry, a glass slide. The samples were recorded at through a simple protocol of semen collection 100x magnification during four minutes, with and to establish basic parameter values for the a random change of the microscope field every evaluation of the reproductive potential of J. ten seconds. Sperm analysis was carried out multidentata populations. with a videomicroscopy system consisting of a phase microscope (Olympus® CX41) and a Materials AND METHODS digital camera (ICAM 1500; Labomed). One hundred and fifty individual cells were tracked Semen collection: Forty-five adult per sample. Each track was followed for one males of J. multidentata (mean standard second divided in seven steps. Sperm dynamic length: 28.83±3.41mm; mean body weight: parameters were analyzed with two softwares. 998 Rev. Biol. Trop. (Int. J. Trop. Biol. ISSN-0034-7744) Vol. 62 (3): 997-1006, September 2014 The ImageJ (NIH, USA) plugin MTrackJ Sperm morphometry: 20µL of sperm (ver.191.1.0, Eric Meijering; www.images- sample were fixed with 2% formaldehyde and cience.org/meijering/software /mtrackj/) was then stained with Coomassie-blue (220% wt/ used to obtain the X and Y coordinates of each vol). The microphotographs were taken at 1 track. On the other hand, each track was ana- 000x magnification using a light microscope lyzed by the Spermtrack IV (Centre for Cell (Olympus® CX31) and a digital camera (Moti- and Molecular Biology, University of Cordoba) cam 2300). The total sperm length (TSL), head to calculate the following kinetic parameters: length (HL) and midpiece length (MPL) were (i) Straight line velocity (VSL) (µm/s): Straight measured using the software Image J (version distance traveled by the spermatozoon from the 1.42q, NIH, USA). A mean value per individual beginning to the end of its track over time, (ii) was calculated for each parameter (20 sperma- Curvilinear velocity (VCL) (µm/s): Length of tozoa per male). the spermatozoon track over measurement time In order to corroborate the spermatozoa and (iii) Linearity (LIN): The quotient between lengths, gonopodium of five males were fixed VSL and VCL as an adimensional value that with 2% of glutaraldehyde and 4% formalde- indicates the grade of straightness of a track hyde in 0.1M cacodylate buffer for 2h, and then post-fixed with osmium tetroxide at 1% (expressed in percentage), where values near in the same buffer, dehydrated and embed- 100% represent a linear movement and values ded in Araldite. Thin sections were cut with near 0%, a more erratic path. a diamond knife on a JEOL JUM-7 ultra- microtome, mounted on nickel grids, con- Viability and sperm count: Fifteen min-
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