The effect of ions and cryoprotectants upon sperm motility and fertilization success in the loach Misgurnus anguillicaudatus
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.
Section snippets
Gamete sampling and evaluation of sperm motility
Wild parental fish were obtained from Iwamizawa city (Hokkaido Island, Japan) during the spawning season (June–August). Gamete maturation was achieved as described in our previous work (Fujimoto et al., 2004) with a single dose of human chorionic gonadotropin (Aska Pharmaceuticals, Tokyo — 100 I.U. per male and 500 I.U. per female) injected intraperitoneally. After 10 to 12 h at 27 °C, fish were anesthetized in 2-phenoxyethanol solution (0.1%) and oocytes were collected in 2 mL tubes by stripping
Sperm parameters
Immotile sperm, and sperm exhibiting progressive motility and non-progressive motility are shown in Fig. 1. The percentage of progressive cells was 80.9% for intact fresh sperm, 76.3% for centrifuged fresh sperm and 72.3% for sperm treated with 30 mM NaCl. None of these values were significantly different (P > 0.05). Potassium maintained sperm quality at increasing concentrations. Average progressive motility was 74, 75%, 82% and 76% for the respective concentrations of potassium at 1.25, 2.5, 5
Discussion
In the present study, we demonstrate that sperm motility parameters were affected by the addition of calcium, magnesium and DMSO. Calcium has previously been reported to exhibit negative effects upon the sperm of some fish species (Christen et al., 1987, Cosson et al., 1991). Divalent cations such as calcium and magnesium have the ability to induce membrane fusion or adhesion (Ahkong et al., 1975, Ohki and Ohshima, 1985, Takeda and Kasamo, 2002). Consequently, the reason for sperm aggregation
Acknowledgments
This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) for Scientific Research (B) (No. 18380108), from the Japanese Society for Promotion of Science (JSPS) to K.A., and for Young Scientists (B) (No. 18780138) from JSPS to T.F.
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