DNA fragmentation and membrane damage of bocachico Prochilodus magdalenae (Ostariophysi: Prochilodontidae) sperm following cryopreservation with dimethylsulfoxide and glucose

Abstract

The endangered bocachico Prochilodus magdalenae is a native freshwater fish of Colombia, the most captured species locally and one of the most important species for ex-situ conservation (germplasm banks). The aim of this study was to examine the effect of three concentrations of Dimethylsulfoxide (DMSO) (5%, 10%, 15%) and three of glucose (305, 333, 361 mM) in the extender on spermatic DNA fragmentation (F-DNA) (by Halomax®, Chromatin dispersion) and membrane damage (D-Me) (by eosin-nigrosin staining). After assessment of sperm quality by computer analysis of motility, one part of semen from males was diluted separately with three parts of extender and filled into 0.5 ml straws. Freezing was carried out in liquid nitrogen vapor dry shipper for 30 minutes and thawed at 60°C for 8 seconds in a water bath and evaluated for the percentage of cells found with F-DNA and D-Me. The results demonstrated that cryopreservation causes greater F-DNA (13.62 ± 1.6% to 28.91 ± 3.25) and D-Me (24.27 ± 1.1% to 58.33 ± 2.81%) when compared with pre-freezing semen (PFS) (6.71 ± 1.54% and 2.34 ± 0.5%, respectively for F-DNA and D-Me). A significant interaction was found between DMSO and glucose concentration in this experiment. Use of extender: 10% DMSO + 305 mM glucose + 12% chicken egg yolk and, 10% DMSO + 333 mM glucose + 12% chicken egg yolk, allow for lower F-DNA and D-Me during cryopreservation of bocachico semen. A high correlation between F-DNA and D-Me was found (r = 0.771).O curimba Prochilodus magdalenae, é uma espécie nativa de água doce da Colômbia ameaçada de extinção, sendo a mais capturada localmente e uma das mais importantes para a conservação ex-situ (bancos de germoplasma). O objetivo deste estudo foi avaliar o efeito de três concentrações de dimetilsulfóxido (DMSO) (5%, 10%, 15%) e três de glicose (305, 333, 361 mM) no diluente sobre a fragmentação do ADN espermático (F-DNA) (através de Halomax®, dispersão da cromatina) e danos em membrana (D-Me) (através da coloração eosina-nigrosina). Depois de avaliar a qualidade espermática por análise computacional da mobilidade, uma parte do sêmen dos machos foi diluída separadamente com três partes do diluente e colocado dentro de canudos de 0.5 ml. O congelamento foi feito em tanque de vapores secos de nitrogênio líquido por 30 minutos e descongelado a 60°C por 8 segundos em banho de água e avaliada a percentagem de células com F-DNA e D-Me. Os resultados demonstraram que a criopreservação causa grande F-DNA (13.62 ± 1.6% até 28.91 ± 3.25) e D-Me (24.27 ± 1.1% até 58.33 ± 2.81%) quando comparada com o sêmen pré-congelamento (PFS) (6.71 ± 1.54% e 2.34 ± 0.5%, respectivamente para F-DNA e D-Me). Uma interação significativa foi encontrada entre a concentração de DMSO e glicose neste experimento. O uso dos diluentes 10% DMSO + 305 mM glicose + 12% gema de ovo de galinha e 10% DMSO + 333 mM glicose + 12% gema de ovo de galinha permitem obter a menor F-DNA e D-Me durante a criopreservação do sêmen de curimba. Uma alta correlação entre F-DNA e D-Me foi encontrada (r = 0.771).

Cryopreservation of Prochilodus brevis semen: freezing media and thawing rates

“…This is the first study reporting data on the vitality of P. brevis sperm. Martinez et al (2012) reported higher values of live spermatozoa from frozen sperm of P. magdalenae, a species belonging to the same genus of the species under study.…”

Crioconservación de semen de dorada Brycon moorei con dimetilsulfóxido

“…Varios estudios han encontrado alta correlación entre el daño mitocondrial y la movilidad total (Fraser & Strzezek, 2007;Martínez et al, 2012;Padilla, 2014). La funcionalidad de la mitocondria juega un papel importante en la activación espermática, ya que suministra la energía en forma de ATP necesaria el movimiento del espermatozoide y la duración del mismo (Medina-Robles et al, 2005).…”

Insemination of bocachico fish (Prochilodus magdalenae) with fresh or cryopreserved semen: effect of spermatozoa/oocyte ratio

“…Additional advantages include broadening artificial fertilization processes in aquaculture (Watson and Holt, 2001) as well as increasing the availability of semen during periods of low sperm production observed in captive fish. Reports on advances in the development of protocols for bocachico semen cryopreservation include: a study on the effect of glucose concentration on sperm motility (Martínez et al, 2011), another study on the use of dimethylacetamide as cryoprotectant (Atencio-García et al, 2013), a further study into the use of different concentrations of dimethyl sulfoxide (DMSO 5, 10, 15%) and glucose (5.5, 6.0, 6.5%) as cryoprotectants (Martínez et al, 2012a), an assessment of membrane damage and DNA fragmentation of bocachico spermatozoa caused by cryopreservation with DMSO and glucose (Martínez et al, 2012b), and a report on the effects of freezing and thawing rates on sperm motility (Martínez et al, 2013). In spite of this, there is little information concerning effective spermatozoa/oocyte ratios for bocachico using fresh and cryopreserved semen to improve the efficiency of semen usage in artificial reproduction processes.…”