Purification and partial characterization of vitellogenin from spotted wolffish (Anarhichas minor) and development of an enzyme-linked immunosorbent assay for the determination of gender and sexual maturity

Abstract

Vitellogenin (VTG) from spotted wolffish, Anarhichas minor, a candidate species for cold-water marine aquaculture, was purified by MgCl₂/EDTA precipitation followed by a two-step chromatographic procedure. VTG had an apparent molecular mass of 470 kDa, as determined by gel filtration, and an amino acid composition similar to those of other teleosts. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified VTG revealed a major band with a relative molecular weight of 166 kDa and some minor bands. Spotted wolffish VTG (sw-VTG) is relatively robust to in vitro degradation, as shown when samples of purified VTG and plasma from mature females subjected to various storage conditions or multiple freeze/thaw cycles were analyzed by Western blot. We developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) using an antibody against Atlantic wolffish (Anarhichas lupus) VTG and purified sw-VTG. The ELISA had a detection limit of 6.7 ng/ml and a working range of 16.2-787.5 ng/ml, with intra- and inter-assay coefficients of variation ranging from 1.5 to 7.3 % and 7.1 to 14.3 %, respectively. The assay could distinguish males from immature females and discriminate maturing females at different stage of oocyte development. These results suggest that the sw-VTG ELISA would be useful in spotted wolffish aquaculture to determine sex and monitor female maturation.

The status of spotted wolffish, Anarhichas minor: A commercially ready species for U.S. marine aquaculture?

“…Accordingly, males do not display normal spawning behavior and females release unfertilized eggs, so fertilization must be conducted in vitro (Foss et al, 2004;Kime & Tveiten, 2005;Le François et al, 2010). Maltais et al (2013) developed an assay for the determination of gender and sexual maturity status, whereas monitored levels of steroid hormones during two successive spawning cycles in A. minor and A. lupus. However, in addition to presumed internal fertilization, several particularities of spotted wolffish reproduction make this species unlike most other commonly cultivated species according to Kime and Tveiten (2005): a short supply of sperm that is motile at extraction but inactivated at contact with seawater, spermatozoa with slow but lasting motility (up to 2 days), and slightly diluted semen.…”

Spotted Wolffish Broodstock Management and Egg Production: Retrospective, Current Status, and Research Priorities

“…The production of all-male stocks should, according to these authors, be considered. In this context, the findings of Maltais et al [24] that used purified vitellogenin to develop an indirect competitive ELISA for the determination of gender and sexual maturity of females is of interest, although sex determination would need to be applied earlier in development. One study in the wild mentioned that males grew more in length than females post-maturation [20].…”

Development and validation of a competitive hybrid ELISA for Seriola lalandi Vitellogenin

“…In teleosts, Vtg levels in the plasma directly indicate the females' reproductive stage (Mañanós, Nuñez, Zanuy, Carrillo, & Le Menn, 1994;Mañanós, Zanuy, Le Menn, Carrillo, & Núñez, 1994;Wallace, 1985). Therefore, Vtg levels in plasma can be used to determine sex and maturational stages in females Maltais, Dupont-Cyr, Roy, & François, 2014). Males are also capable of producing Vtg if exposed to oestrogenic compounds .…”

“…The first Vtg ELISA was developed in 1984 as a method for sexing salmonids (Gordon, Owen, Ternan, & Hildebrand, 1984). Since then, Vtg ELISA has been widely used for assessing the maturational stages in different fish species Maltais et al, 2014;Mosconi, Carnevali, Carletta, Nabissi, & Polzonetti-Magni, 1998;Susca, Corriero, Bridges, & Metrio, 2001).…”

“…Using Oreochromis niloticus Vtg as a standard, the levels of circulating Vtg in seven different cichlid species were determined (Ndiaye et al, 2006). The cross-reactivity between Vtg from different species has also been used to develop hybrid ELISAs, where a heterologous antibody is used against a homologous Vtg standard (Carnevali & Belvedere, 1991;Maltais et al, 2014;Maltais, Roy, & Couillard, 2010;Mylchreest, Snajdr, Korte, & Ankley, 2003;Vega-López et al, 2006). In the present study, we evaluated the use of homologous antibodies developed against synthetic peptides (anti-S. lalandi Vtg antiserum) compared with heterologous antibodies developed against the whole Vtg molecule (anti-M. cephalus LvAa/Ab antiserum) in order to develop a competitive ELISA for S. lalandi Vtg.…”