Methyl glycol, methanol and DMSO effects on post-thaw motility, velocities, membrane integrity and mitochondrial function of Brycon orbignyanus and Prochilodus lineatus (Characiformes) sperm

Abstract

The aim of this study was to use more accurate techniques to investigate the effects of cryoprotectants (CPAs) and extenders on post-thaw sperm quality of Brycon orbignyanus and Prochilodus lineatus. Six freezing media comprising the combination of three CPAs (DMSO, methanol and methyl glycol) and two extenders (BTS and glucose) were used. Sperm was diluted in each medium, loaded into 0.5-mL straws, frozen in a nitrogen vapor vessel (dry-shipper), and stored in liquid nitrogen at -196 °C. Post-thaw sperm motility rate and velocities (curvilinear = VCL; straight line = VSL; average path = VAP) were evaluated using a computer-assisted sperm analyzer. Membrane integrity and mitochondrial function were determined using fluorochromes. Post-thaw quality was considered high when samples presented the following minimum values: 60 % motile sperm, 140 µm/s of VCL, 50 % intact sperm membrane and 50 % mitochondrial function integrity. High post-thaw quality was observed in B. orbignyanus sperm frozen in BTS-methyl glycol and in P. lineatus sperm frozen in BTS-methyl glycol, glucose-methyl glycol and glucose-methanol. All samples frozen in DMSO yielded low quality. The presence of ions in the BTS extender affected post-thaw sperm quality positively in B. orbignyanus and negatively in P. lineatus. Methyl glycol was the most suitable CPA for both fish species, leading to a good protection of cell membrane, mitochondrial function and motility apparatus during the cryopreservation process. For an improved protection, B. orbignyanus sperm should be frozen in an ionic freezing medium.

Effects of cryopreservation on mitochondria of fish spermatozoa

“…Spermatozoa are protected by various antioxidants and antioxidant enzymes, found in the seminal plasma or in the spermatozoa itself, which prevent oxidative damage (Kim & Parthasarathy 1998;Lahnsteiner & Mansour 2010;Li et al 2010;Shaliutina et al 2013;). An antioxidant that reduces oxidative stress and improves sperm motility could be useful in cryopreservation protocols (Lahnsteiner et al 2011;Gazo et al 2014 Recent studies demonstrate that supplementation of cryopreservation extenders with antioxidants provides a cryoprotective effect on bull, ram, goat, boar, canine, human and fish sperm quality, thus improving semen parameters such as sperm motility, membrane integrity and ΔΨ m after thawing (Bucak et al 2010;Cabrita et al 2011;Mart ınez-P aramo et al 2012;Figueroa et al 2015;Viveiros et al 2015).…”

Cryopreservation of sperm in annual fish Austrolebias minuano (Cyprinodontiformes; Rivulidae)

“…However, given the variation in the biochemical composition of sperm, individualized species‐specific cryopreservation protocols are necessary for each species (Viveiros et al, ). An efficient and practical cryopreservation protocol for sperm needs to take into account and standardize factors like type and concentration of cryoprotectants, diluents, osmolarity, freezing and thawing temperatures of spermatozoa and duration of each processing step.…”

“…Dimethyl sulfoxide (DMSO), glycerol and methyl glycol (MG) are common intracellular cryoprotectants used for cryopreservation of fish spermatozoa (Viveiros et al, ). Indeed, alcohol‐based cryoprotectants are widely used for cryopreservation of sperm from freshwater fish: Piracanjuba ( Brycon orbignyanus ; Perry et al, ), Tambaqui ( Colossoma macropomum ; Garcia et al, ; Varela Junior et al, ), Curimba ( Prochilodus lineatus ; Miliorini et al, ; Viveiros et al, ), Pacu ( Piaractus mesopotamicus ; Pires et al, ).…”

“…DMSO shows cryopreservative capabilities in freezing of fish semen due to its high solubility in an aqueous medium, easily penetrating the plasma membrane, and engaging the free hydroxyl groups to inhibit the formation of reactive oxygen species (Viveiros et al, ). Glycerol is another common agent used for cryopreservation of male gametes (Ahn, Park, & Lim, ), although certain factors, such as like its high molecular weight, viscosity and impermeability, make it less favourable for use as a cryoprotectant (Miliorini et al, ).…”

“…Glycerol is another common agent used for cryopreservation of male gametes (Ahn, Park, & Lim, ), although certain factors, such as like its high molecular weight, viscosity and impermeability, make it less favourable for use as a cryoprotectant (Miliorini et al, ). A number of studies indicate that MG is an ideal cryoprotectant for fish spermatozoa, based on investigations in Brycon insignis (Viveiros et al, ), Piaractus brachypomus (Ramirez‐Merlano, Velasco‐Santamaría, Medina‐Robles, & Cruz‐Casallas, ), Piaractus mesopotamicus and Piaractus lineatus (Viveiros et al, ), Colossoma macropomum (Carneiro, Azevedo, Santos, & Maria, ) and Steindachneridion scriptum (Ribeiro et al, ).…”