The effect of ions and cryoprotectants upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus

Abstract

The solutions commonly used to dilute or cryopreserve sperm are commonly composed of salts, buffers and cryoprotectants, which may affect gametes and subsequent fertilization success. Here, we have evaluated the effects of several cryoprotectants (methanol; MeOH, dimethyl sulfoxide; DMSO and dimethyl acetamide; DMA at concentrations of 0.25, 0.5 and 1%) and different ions (potassium, calcium and magnesium at concentrations of 1.25, 2.5, 5.0 and 10 mM) as sperm diluents upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus sperm. Our results demonstrated that DMSO (at 1%) decreased sperm motility while calcium and magnesium ions (from 2.5 mM) induced sperm aggregation and reduced sperm motility. Reduced fertilization rates were observed with potassium (from 1.25 mM), calcium (at 10 mM), magnesium (at 10 mM), DMA (at 1%), and DMSO (at 1%). We conclude that specific ions and cryoprotectants, and their relative concentrations caused effect upon loach gametes. These data are important to consider for the preparation of sperm diluents and activating solutions in order to manage gamete quality for artificial propagation.

Introduction

The application of in vitro fertilization (IVF) for species of fish is important for the reproductive manipulation of species that do not spawn spontaneously under cultured conditions. Hatchery procedures such as artificial incubation and larval rearing, in combination with IVF, may greatly increase hatching rate and the survival of resultant juveniles (Ciereszko et al., 2000, Woynarovich and Horváth, 1980). The potential benefits are highly relevant since IVF would optimize the large-scale production of seeds for aquaculture. Furthermore, IVF permits the application of biotechnology related to chromosome set manipulation (Arai, 2001) and the utilization of cryopreserved sperm (Billard and Zhang, 2001).

The general hatchery procedure for IVF in fish involves mixing sperm with the oocyte mass, followed by activation using water (the so-called ‘dry method’). Following activation, spermatozoa become motile and enter the oocyte via the micropyle in order to achieve fertilization (Hart, 1990, Linhart et al., 1995). Following this, the water volume is increased and the fertilized eggs are incubated in well-aerated water in order to promote hatching (Legendre et al., 1996, Woynarovich and Horváth, 1980). The external media used to activate sperm, may affect important sperm characteristics (Litvak and Trippel, 1998, Morisawa et al., 1983, Wojtczak et al., 2007, Yoshida and Nomura, 1972), fertilization success (Ketola et al., 1988, Saad and Billard, 1987) and subsequent embryo development (Gonzal et al., 1987, Molokwu and Okpokwasili, 2002, Silva et al., 2003). During fish IVF, the ionic components of the fertilization media are influenced by the water source, ovarian fluid and the sperm.

Fish sperm is commonly diluted in physiological media (known as ‘extenders’), as this optimizes sperm usage (Linhart et al., 1987, Rodina et al., 2004, Scott and Baynes, 1980). The composition of extenders must be carefully determined in order to optimize sperm viability and fertilization success. Another method frequently employed in fish IVF is to use cryopreserved sperm. In such cases, diluents often contain cryoprotectants such as methanol and DMSO that may also exert detrimental effects upon gametes (Kopeika et al., 2003). Sperm cryopreservation is particularly interesting for genebanking in the loach as individuals such as natural clones, polyploids and hybrids are important for academic research and aquaculture (Arai, 2001).

In our previous studies, we succeeded in cryopreserving loach sperm with high rates of sperm motility and subsequent fertilization (Yasui et al., 2008, Yasui et al., 2009). However, we observed that fertilization rates were severely reduced if cryopreserved sperm were not sufficiently diluted (unpublished data). This fact suggests that some components from sperm diluents may be toxic at a given concentration and must be diluted sufficiently to improve fertilization. However, although we confirmed the importance of sperm dilution in order to optimize fertilization, it is still necessary to identify which components are toxic and determine safe concentrations or even eliminate from sperm diluents. Increasing understanding of these characteristics is likely to be applicable in the preparation of sperm diluents and activating solutions, and improvement of the management of gametes for artificial propagation. In the loach, many kinds of solutions have been used to dilute sperm including modified Ringer solution (Suzuki et al., 1985), Kurokura's solution (Fujimoto et al., 2008, Yasui et al., 2008, Yasui et al., 2009) and NaCl 0.9% (unpublished data). For cryopreservation, we previously used potassium solution (Yasui et al., 2008, Yasui et al., 2009) and a sodium-based solution (Yasui et al., 2010), both containing methanol as the cryoprotectant. Most of these solutions were empirically produced or based on previous studies of other fish species. However, we do not know which component is necessary, or if it presents potentially detrimental effects. Nevertheless, it is important to produce such solutions that are appropriate for the loach in order to optimize sperm maintenance and fertilization success.

Consequently, the aim of the present study was to evaluate the toxicity of some components commonly used in sperm diluents that may affect both sperm and subsequent fertilization success in the loach. This study will allow us to prepare non-toxic extenders, cryo-solutions, and to manage sperm dilution ratios in order to maximize fertilization rates in the aquaculture setting.