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Seminal and Spermatic Characteristics of Fresh Semen and the Effects of Sperm Cooling in Steindachneridion Melanodermatum (Garavello, 2005)

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Seminal and Spermatic Characteristics of Fresh Semen and the Effects of Sperm Cooling in Steindachneridion Melanodermatum (Garavello, 2005) DOI: 10.5433/1679-0359.2015v36n6Supl2p4493 Seminal and spermatic characteristics of fresh semen and the effects of sperm cooling in Steindachneridion melanodermatum (Garavello, 2005) Características seminais e espermáticas e os efeitos do resfriamento de esperma do Steindachneridion melanodermatum (Garavello, 2005) sobre os parâmetros espermáticos. Ronan Maciel Marcos1; Giovano Neumann2*; Cesar Pereira Rebechi de Toledo2; João Marcos Sena2; Gilmar Baumgartner3; Robie Allan Bombardelli3 Abstract This study describes the seminal and spermatic characteristics of fresh semen of Steindachneridion melanodermatum and investigates the effects of dilution, temperature, and storage period on its spermatic parameters. Sperm samples were collected from nine hormonally-induced males. The following parameters in fresh sperm were analyzed: seminal plasma osmolality (OSM), seminal pH, sperm motility (MOT), sperm velocity (SV) (including sperm curvilinear velocity (VCL), sperm straight-line velocity (VSL), and sperm average path velocity (VAP)), total time of sperm motility (TEMP), sperm concentration (CONC), and index of sperm normality (NORM). Sperm samples from each male were diluted in a solution containing 5% fructose and 5% powdered milk, and stored at 10°C and 25°C. The same was carried out for sperm samples not subjected to dilution. From these samples, MOT, VCL, VSL, VAP, SV, and TEMP were measured after 0 h, 5 h, 9 h, 18 h, 27 h, 36 h, 45 h, and 54 h. Males released 11.74 ± 5.38 mL of sperm, with an osmolality of 258.78 ± 29.36 mOsm.kg-1 and pH of 7.11 ± 0.31. The sperm presented a MOT of 99.86 ± 0.31% at a concentration of 1.03 × 1010 ± 3.65 × 109 spermatozoa.mL-1 with VCL of 185.58 ± 14.11 μm.s-1, VSL of 49.15 ± 4.66 μm.s-1, VAP of 87.02 ± 4.13 μm.s-1, SV of 106.52 ± 4.45 μm.s-1, TEMP of 79.31 ± 5.62 s, NORM of 75.81 ± 5.71%. The results indicate that sperm motility, sperm velocity, and total time of sperm activation were affected by dilution, storage temperature, and storage period (p < 0.05). Procedures for semen storage should be performed with undiluted sperm cooled at 10°C, or kept undiluted at 25°C for up to 27 h. Key words: CASA, endemic, sperm characterization, sperm motility, sperm storage, sperm velocity Resumo O objetivo deste estudo foi descrever os parâmetros seminais e espermáticos no sêmen fresco do Steindachneridion melanodermatum e os efeitos da diluição, da temperatura e do tempo de estocagem do sêmen sobre os parâmetros espermáticos. Foi coletado o sêmen de nove machos, induzidos hormonalmente. No sêmen fresco foram avaliados os parâmetros: osmolaridade do plasma seminal (OSM), pH seminal (pH), motilidade (MOT) e velocidades espermáticas (VCL, VLR, VMD, VE), 1 Mestre em Recursos Pesqueiros e Engenharia de Pesca, Professor Adjunto da Universidade Federal da Fronteira Sul, UFFS, Laranjeiras do Sul, PR, Brasil. E-mail: [email protected] 2 Mestres em Recursos Pesqueiros e Engenharia de Pesca, discentes, Universidade Estadual do Oeste do Paraná, UNIOESTE, Toledo, PR. E-mail: [email protected]; [email protected]; [email protected] 3 Professores Doutores Adjuntos da Universidade Estadual do Oeste do Paraná, UNIOESTE, Toledo, PR. E-mail: gilmar_baum@ yahoo.com.br; [email protected] * Author for correspondence Recebido para publicação 28/10/14 Aprovado em 22/05/15 4493 Semina: Ciências Agrárias, Londrina, v. 36, n. 6, suplemento 2, p. 4493-4506, 2015 Marcos, R. M. et al. tempo total de motilidade espermática (TEMP), concentração espermática e índice de normalidade espermática (NORM). De cada macho, amostras de sêmen que foram diluídas em solução contendo 5% de frutose e 5% de leite em pó e, armazenadas a 10 e 25ºC. O mesmo foi feito para amostras de sêmen não submetidas à diluição. Destas amostras, as MOT, VCL, VLR, VMD, VE, TEMP foram mensuradas após 0, 5, 9, 18, 27, 36, 45, 54 horas. Os machos liberaram 11,74 ± 5,38 mL de sêmen com 258,78 ± 29,36mOsm.kg-1 e pH de 7,11 ± 0,31. O sêmen apresentou 99,86 ± 0,31% de MOT, com 1,03x1010 ± 3,65x109 espermatozódes.mL-1 e, 185,58 ± 14,11μm.s-1 para VCL, 49,15 ± 4,66μm.s-1 para VLR, 87,02 ± 4,13 μm.s-1 para VMD, 106,52 ± 4,45μm.s-1 para VE, 79,31 ± 5,62s para TEMP e 75,81 ± 5,71% de NORM. A motilidade, as velocidades e o tempo total de motilidade espermática foram influenciados (p<0,05) pela diluição, pelas temperaturas e tempos de armazenamento. A estocagem do sêmen poderá ser realizado sem diluição e resfriado a 10oC ou sem diluição, a temperatura de 25oC, pelo período de até 27 horas. Palavras-chave: Caracterização seminal, CASA, endêmico, estocagem de sêmen, motilidade espermática, velocidade espermatica Introduction conservation. The few available reports provide superficial data regarding sperm release and some The Iguaçu sorubim, Steindachneridion sperm traits (LUDWIG et al., 2005; SANT’ANNA, melanodermatum (GARAVELLO, 2005), is a South 2009). American catfish, endemic to the Iguaçu River basin (FROESE; PAULY, 2014). It is carnivorous Since seminal and spermatic parameters allow (ZANIBONI-FILHO et al., 2004), and reaches large the rational utilization of high-quality gametes sizes (length < 532 mm) (GARAVELLO, 2005). and superior broodstock identification (COWARD This is a migratory species, which is subject to et al., 2002), they enable the development of potential extinction (SUZUKI et al., 2003), and due efficient broodstock management by using adequate to dams on the Iguaçu River, its current distribution inseminating dosages (BOMBARDELLI et al., is restricted to the stretches between hydroelectric 2006; STREIT JÚNIOR et al., 2008), cryopreserved plants (GARAVELLO, 2005). These facts and the semen viability in artificial fertilization processes lack of information regarding Steindachneridion (SUQUET et al., 2000), and technology development reproductive biology (HONJI et al., 2012) suggest for the formation of genetic banks (ZANIBONI- advanced research on their genetic variability FILHO; SCHULZ, 2003) directed to biodiversity (MATOSO et al., 2011) and artificial reproduction. conservation or genetic improvement programs. Moreover, due to their growth characteristics and The development of methods focusing on the meat quality, research has been conducted to assess conservation of male gametes in Neotropical fish the nutritional aspects (LEWANDOWSKI et al., has been intensively investigated (VIVEIROS et 2013) and its potential for rearing in net cages al., 2010), as the gametes quickly become unviable (FEIDEN et al., 2013). after ovulation or spermiation (RIZZO et al., 2003). The development of breeding techniques is Besides cryopreservation by freezing, semen the first step to enable artificial fish propagation storage techniques at low temperatures might favor (ROMAGOSA et al., 2010) and the formation of gamete preservation over short periods (JENKINS; either in vivo or in vitro genetic banks (ZANIBONI- TIERSCH, 1997). These procedures might assure: FILHO; SCHULZ, 2003). However, there is no artificial breeding in cases of asynchronous gamete information regarding the reproductive features production (SANCHES et al., 2011a), commercial of S. melanodermatum in captivity, or efficient trade of fresh semen between laboratories and biotechnology tools for gamete manipulation and companies, or maintainance of viable gametes 4494 Semina: Ciências Agrárias, Londrina, v. 36, n. 6, suplemento 2, p. 4493-4506, 2015 Seminal and spermatic characteristics of fresh semen and the effects of sperm cooling in Steindachneridion ... from distant collection sites for further laboratory Semen was collected after 260 hours-degree (10 procedures in genetic conservation programs. h; water temperature 26°C) by massaging the coelomatic region from head to tail (LUDWIG et al., This study describe the seminal and spermatic 2005) after anesthetizing the fish by immersion in characteristics on fresh semen and investigated the water containing 75 mg benzocaine.L-1 (OKAMURA effects of dilution, temperature, and storage period et al., 2010). The first sperm drops were discarded on the sperm parameters of Steindachneridion to avoid urine or feces contamination (POUPARD melanodermatum semen. These data will provide et al., 1998). Released semen volume was measured information supporting the implementation and for each specimen using graduated assay tubes with maintenance of gene banks and breeding programs an accuracy of 0.1 mL. focused on species conservation and aquaculture. After collection, semen samples of each male were evaluated to determine seminal and sperm Material and Methods parameters. We evaluated sperm for: plasma The experiment was carried out during October osmolality (OSM), seminal pH (pH), sperm motility 2010 at the Experimental Station of Ichthyological (MOT), curvilinear velocity (VCL), straight-line Studies of the Energetic Company of Paraná State, velocity (VSL), average path velocity (VAP), total Hydroelectric Power Plant Governador Ney Braga time of sperm motility (TEMP), sperm concentration (EEEI - Copel) in Iguaçu Reserve, Paraná, Brazil, (CONC), and index of morphological normality of and in the Laboratory of Reproductive Technology spermatozoa (NORM). to Aquatic Animals (LATRAAC) at the University To determine OSM, 2.5 mL of semen was of Western Paraná State, Toledo, Paraná, Brazil. centrifuged at 3,500 rpm for 10 min. Then, the The broodstock from EEEI - Copel was seminal plasma was separated and stored in maintained through the year in ground tanks Eppendorf tubes in the freezer at -20°C. The with concrete ledges and sandy bottoms. Water osmolality was established by analyzing the samples was pumped from the Salto Segredo Reservoir using a cryoscopic method with an osmometer (PLZ, in the Iguaçu River to replace water lost through model PLZ-1000) (ALAVI et al., 2010). Seminal evaporation and infiltration. Animals were fed pH was determined by a colorimetric method using commercial extruded food containing 32% raw litmus paper (TESSARO et al., 2012). One drop protein at a rate of 3% of the fish weight per day. Live of semen was placed on the paper and the color food (small fish) was also given to the broodstock.
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