Vitamin E and reduced glutathione in Prochilodus lineatus (curimba) semen cryopreservation (Characiformes: Prochilodontidae)

Abstract

This study investigated the addition of antioxidants vitamin E and reduced glutathione on curimba (Prochilodus lineatus) semen cryopreservation and compared sodium bicarbonate solution and distilled water as activators. The experiment was conducted at the environmental station of CEMIG, in Itutinga-MG, Brazil, between December/2009 and January/2010. Semen samples (n = 7) with semen motility above 80% were diluted in cryoprotectant solutions composed of 10% methanol, 15% lactose and containing different concentrations of antioxidants: 50 (VE50), 100 (VE100) and 250 (VE250) µM of vitamin E, and 0.5 (RG0.5), 1.0 (RG1.0) and 1.5 (RG1.5) mM of reduced glutathione. A solution without antioxidants was used as a control. The semen was diluted at a ratio of 1:4 (100 ìL semen:400 μL cryoprotectant solution). The toxicity of the solutions was evaluated by investigating semen motility after 10 min in the solution. The rest of the diluted semen was placed into 0.5 mL straws maintained in nitrogen vapour for 24 hours and packed into a nitrogen liquid cylinder for four days. The samples were thawed in a water bath at 60°C for 8 s and the rate (%) and duration (s) of semen activation with distilled water or sodium bicarbonate was evaluated. In the toxicity test, we found that vitamin E and reduced glutathione were not toxic to curimba semen at any of the tested concentrations (P>0.05). The duration of motility was longer (P<0.05) in semen activated with sodium bicarbonate 1% (163 ± 11 s), which was considered the best activator for semen under these conditions. No significant differences were found between the cryoprotectant solutions used after freezing (P>0.05). Thus, the antioxidants vitamin E and reduced glutathione did not improve the quality of cryopreserved curimba semen, but they did not cause toxic effects to the semen in natura and they did not decrease its quality during cryopreservation.Este estudo avaliou a adição de antioxidantes vitamina E e glutationa reduzida no sêmen criopreservado de curimba (Prochilodus lineatus) e comparou solução de bicarbonato de sódio e água destilada como ativadores. O experimento foi conduzido na estação ambiental da CEMIG, em Itutinga-MG, entre Dezembro/2009 e Janeiro/2010. Sêmen de sete animais, com motilidade espermática acima de 80%, foi diluído em soluções crioprotetoras compostas por metanol 10% e lactose 15% em diferentes concentrações de antioxidantes: 50 (VE50), 100 (VE100) e 250 (VE250) µM de vitamina E, 0,5 (RG5.5), 1,0 (RG1.0) e 1,5 (RG1.5) mM glutationa reduzida e uma solução controle sem antioxidante. O sêmen foi diluído na proporção de 1:4 (100 µL de sêmen: 400 µL de solução crioprotetora). A toxicidade das soluções foi avaliada pela motilidade espermática após de 10 minutos em solução. O restante do sêmen diluído foi armazenado em palhetas de 0,5 mL mantidos em vapor de nitrogênio por 24 horas e estocado em cilindro de nitrogênio líquido por quatro dias. As amostras foram descongeladas em banho-maria a 60°C por 8 segundos e avaliada a taxa (%) e duração (s) pela ativaçã...

Effect of Vitamins and Amino Acids as Cryoprotectant Solution Supplementation for the Cryopreservation of Tambaqui Semen (Colossoma macropomum)

“…Apesar de já haver registros sobre o sucesso da utilização de antioxidantes na concentração de 1 mM na criopreservação seminal de teleósteo [14], nas condições empregadas nesse estudo, os resultados encontrados nos tratamentos suplementados com as substâncias antioxidantes foram, de modo geral, similares aos encontrados para o tratamento não suplementado. O mesmo foi relatado por outros autores, que não observaram melhora nos parâmetros cinéticos e morfológicos do sêmen criopreservado após suplementação com antioxidantes, nas espécies Dicentrarchus labrax, Sparus aurata e Prochilodus lineatus [6,21].…”

Effects of cooling rates on the quality of Prochilodus lineatus (Valenciennes, 1836) sperm

“…To determine spermatic concentrations, a 10 μl aliquot of fresh sperm of each fish was collected, diluted in 990 μl of a formaldehyde citrate solution (2.9 g sodium citrate, 4 ml formaldehyde 35% made up with distilled water to 100 ml) (Andrade et al, ; Felizardo et al, ; Paula et al, ; Pinheiro et al, ). After dilution, 10 µl of the diluted samples was mixed with 990 µl of the formaldehyde citrate solution, resulting in a final dilution of 1:10 4 (sperm:formaldehyde citrate).…”

“…The following parameters were evaluated: per cent motility, duration of sperm motility, velocity curvilinear (VCL), velocity average path (VAP), velocity straight line (VAS), straightness (STR), wobble (WOB) and progression (PRO). The sperm motility duration analysis was performed using the ImageJ software, through a complete video of sperm movement until approximately 10% of the sperm in the evaluated field were mobile (Felizardo, Murgas, Navarro, Gonçalves, & Paulino, 2011;Murgas, Miliorini, Franciscatto, & Maria, 2004;Murgas et al, 2007;Paula et al, 2012).…”

“…In recent years, many studies have been carried out regarding P. lineatus semen cryopreservation, and several assessments have contributed with relevant information regarding its cryopreservation and evaluation. Thus, factors such as the use of cryoprotectants and their concentrations, thawing temperatures and time, antioxidant substance use or not, semen storage time and activating solution have been determined (Carvalho et al, 2017;Di Chiacchio, Almeida, Leal, & Viveiros, 2017;Felizardo et al, 2010;Gonçalves, Nascimento, Costa, Leal, & Viveiros, 2013;Miliorini et al, 2011;Murgas, Miliorini, Freitas, & Pereira, 2007;Paula et al, 2012). Miliorini et al (2011), for example, observed that P. lineatus cryopreserved semen samples containing 5% BTS and dimethylsulfoxide presented the lowest percentage of fractured tails and tail stumps.…”

Toxicity of cryoprotectants on Prochilodus lineatus (Valenciennes, 1837) (curimba) embryos in an experimental incubator (Characiformes: Prochilodontidae)

“…Furthermore, it is used as a model in research in the reproductive biotechnology of Brazilian fish (Viveiros et al, 2010b), thanks to its high prolificacy and ease of management. As a migratory species, curimba needs to swim upriver to spawn and this cycle has been drastically affected (Paula et al, 2012). Cryopreservation is a biotechnology for its conservation and a tool for assisted reproduction on a large scale.…”