Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

Abstract

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate;(3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post-thaw sperm motility was achieved with the sperm being frozen at a cooling rate of À5°C min À1 to À30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post-thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L-ascorbic acid, on the other hand, significantly improved the postthaw sperm motility. However, only the addition of 0.6% glycine improved the post-thaw sperm fertilization rate, which was further confirmed by the improvement of the post-thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.

Cryopreservation of strip spawned sperm using non-programmable freezing technique in the blue musselMytilus galloprovincialis

“…The addition of sugars in cryoprotective medium is one of the key strategies in improving sperm qualities and has been applied to various species (Lyons et al 2005;Naing, Wahid, Azam, Rosnina, Zuki, Kazhal, Bukar, Thein, Kyaw & San 2010;G omez-Fern andez et al 2012;, 2016. In general, sugar could (1) stabilize the membrane during freezing; and (2) be utilized as an energy source where glucose and trehalose have been found to enhance the post-thaw sperm quality.…”

“…; Purdy ; Cabrita, Ma, Diogo, Martínez‐Páramo, Sarasquete & Dinis ; Liu, Li et al . , ; Liu, Xu et al . , ), and has been applied in rams (Jafaroghli, Khalili, Farshad & Zamiri ), boars (Gómez‐Fernández, Gómez‐Izquierdo, Tomás, Mocé & Mercado ), black‐lip pearl oysters (Lyons et al .…”

“…Both programmable and non-programmable freezing techniques have been used in sperm cryopreservation. However, the latter has shown the better post-thaw sperm quality than the former in rhesus macaque Macaca mulatta (Si, Lu, He, Ji, Niu, Tan & Ji 2010), human (Talebian, Sadeghi, Sadri-Ardekani, Bolourzadeh & Akhondi 2005) and farmed greenlip abalone Haliotis laevigata Liu, Li, Xu, Robinson & Qin 2016). The non-programmable freezing technique also requires much lower initial investments and can process larger amount of samples than the programmable freezing technique, therefore, being more convenient for application on sites (Adams et al 2008;Zhu et al 2014).…”

“…Researches in livestock and aquatic species have demonstrated that the addition of sugar and/or amino acid is an effective strategy to improve the post-thaw sperm quality (Lyons et al 2005;Purdy 2006;Cabrita, Ma, Diogo, Mart ınez-P aramo, Sarasquete & Dinis 2011;, 2016, and has been applied in rams (Jafaroghli, Khalili, Farshad & Zamiri 2011), boars (G omez-Fern andez, G omez-Izquierdo, Tom as, Moc e & Mercado 2012), black-lip pearl oysters (Lyons et al 2005), farmed greenlip and black-lip abalone .…”

“…Sperm component and organelle integrity is of importance for sperm to perform their functions (Adams, Hessian & Mladenov 2003;Salinas-Flores, Paniagua-Chavez, Jenkins & Tiersch 2005;Zhang et al 2012) and can be evaluated by specific fluorescence dyes. This technique has been applied in various species to provide detailed information for sperm quality improvement in cryopreservation studies (Salinas-Flores et al 2005;Paniagua-Ch avez et al 2006;, 2016.…”