Elsevier

Aquaculture

Volume 434, 20 October 2014, Pages 362-366
Aquaculture

Short communication
Improvement in non-programmable sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata

https://doi.org/10.1016/j.aquaculture.2014.08.033Get rights and content

Highlights

  • Glucose, fructose and galactose showed a similar cryoprotective effect.

  • The addition of 0.6% glycine has improved the post-thaw fertilization rate to 96%.

  • Substantial reduction in sperm to egg ratio is required for post-thaw fertilization.

Abstract

This study assessed the effects of the addition of vitamins (l-ascorbic acid), amino acids (glycine and taurine), and monosaccharides (glucose, fructose and galactose) on sperm cryopreservation using a non-programmable freezing technique in farmed greenlip abalone. The results showed that the addition of taurine, glycine or l-ascorbic acid significantly improved the post-thaw sperm motility, whereas the post-thaw sperm fertilization rates were improved by the addition of glycine or l-ascorbic acid. Flow cytometry analysis demonstrated that the addition of glycine significantly enhanced the post-thaw sperm plasma membrane integrity and acrosome integrity. Results from the investigation on monosaccharides demonstrated that glucose, fructose and galactose had a similar cryoprotective effect, resulting in a similar level of the post-thaw sperm fertilization rate, plasma membrane integrity, mitochondrial membrane potential and acrosome integrity. In this study, the highest post-thaw fertilization rate of 96% was achieved by using the cryoprotective mediums containing 6% DMSO, 1% glucose and 0.6% glycine.

Introduction

In Australia, genetic improvement programs, such as selective breeding and hybridization, have been established to improve the production and maintain the sustainable and competitive long-term development of the abalone aquaculture industry (Li, 2008). Recently, cryopreservation techniques have been developed and have potential for enhancing the efficiency of these programs by overcoming the asynchronous spawning between male and female in greenlip abalone Haliotis laevigata on farm (Liu et al., 2014). In comparison with sperm collected from wild greenlip abalone (Zhu et al., 2014), those collected from farmed broodstock were more sensitive to cryopreservation. To achieve a post-thaw sperm fertilization rate of about 90% in farmed stocks, high sperm to egg ratios of 40,000:1 and 10,000:1 were required in programmable and non-programmable freezing techniques, respectively (unpublished data; Liu et al., 2014). For successful application of this technique in commercial hatchery production or genetic improvement programs, a higher fertilization rate is needed using a lower sperm to egg ratio. One of the aims of this study is to investigate whether higher fertilization rates using a lower sperm to egg ratio can be achieved with the addition of amino acids and vitamins when using the non-programmable freezing technique.

Sugars have been widely used as a part of sperm cryoprotective mediums in livestock and fish species. Sugars are thought to lead to more favourable osmotic pressures for the purpose of inducing sperm dehydration and reducing the incidence of intracellular ice formation, resulting in the maintenance of sperm quality during cryopreservation (Gómez-Fernández et al., 2012, Horváth et al., 2003). Glucose has been shown to play a positive role in sperm cryopreservation using the non-programmable freezing technique in farmed greenlip abalone (Liu et al., 2014). In other studies, fructose and galactose can provide better cryoprotection than glucose in canine (Ponglowhapan et al., 2004) dog (Yildiz et al., 2000) and red deer (Fernández-Santos et al., 2007). The effects of different types of monosaccharides on sperm cryopreservation have not been investigated in abalone and other marine mollusc species. Therefore, this study will investigate the effects of sugars, amino acids and vitamins on the success of cryopreservation for farmed greenlip abalone which may be applicable to other marine molluscs.

Section snippets

Animals and gamete preparation

Three year old farmed animals were provided by the SAM Abalone, Port Lincoln, South Australia (SA) in early October and were transported by air to the Aquatic Sciences Centre, South Australian Research and Development Institute (SARDI), Adelaide, SA. Methods for animal acclimation, spawning induction, and collection of concentrated sperm were described by Liu et al. (2014). Eggs were gently poured through a 300 μm sieve and retained on a 90 μm sieve at the bottom. The collected eggs were gently

Effects of different monosaccharides on post-thaw sperm quality in farmed greenlip abalone

No significant difference in post-thaw sperm fertilization rates was found when sperm was cryopreserved in 6% DMSO plus 1% glucose, galactose or fructose at sperm to egg ratios of 2000:1 and 6000:1 (P > 0.05) (Fig. 1). Although 6% DMSO + 1% glucose produced significantly higher post-thaw sperm fertilization rates than the other two monosaccharides at a sperm to egg ratio of 10,000:1 (P < 0.05), the addition of galactose or fructose also resulted in high fertilization rates of 93% (Fig. 1). Evaluation

Discussion

This study has found that the non-programmable sperm cryopreservation technique in farmed greenlip abalone can be improved by the addition of 0.6% glycine in 6% DMSO + 1% glucose. In addition, when glucose, galactose and fructose were applied at the same final concentration, these three monosaccharides showed a similar cryoprotective effect in farmed greenlip abalone.

The supplementation of amino acids and vitamins in cryoprotective medium can improve the post-thaw sperm quality in livestock and

Acknowledgements

We appreciate the financial support from Australian Seafood Cooperative Research Centre (project 2010/755), and Australian Abalone Growers Association and South Australian Research and Development Institute (SARDI). We thank Mr Tom Hyde and Mr Craig Marshall of SAM Abalone and Mr David Connell of Kangaroo Island Abalone for the provision of abalone broodstock and advice on spawning induction. We also thank Dr Paul Van Ruth and Dr James Paterson for assistance in the usage of flow cytometry.

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