Circannual variation in plasma vitellogenin and gonadotropin II levels in relation to annual ovarian cycle in female mrigal, Cirrhinus mrigala

Two forms (HA I and HA II) of vitellogenin (Vg), the yolk-precursor protein, were purified from the plasma of estradiol-17β (E2)-treated Indian major carp, mrigal (Cirrhinus mrigala), by gel filtration on Ultrogel AcA 34 followed by adsorption chromatography on Hydroxylapatite (HA)-Ultrogel. Native HA I and HA II had molecular weights of 500 kDa and 550 kDa, respectively. The apparent masses of purified HA I and HA II after SDS-PAGE under reducing condition were 75 kDa and 85 kDa, respectively. HA I was found to be lipid rich whereas HA II was phosphorous rich. The co-purified Vg containing both HA I and HA II was used to raise polyclonal antisera (a-Vg) and its specificity was assessed by Western blot analysis. In a double immunodiffusion test, the plasma of vitellogenic females and E2-treated males as well as crude egg yolk protein (CYP), cross-reacted with a-Vg giving two precipitin lines in each case, thereby indicating the presence of two forms of Vg. HA I and HA II each gave single precipitin lines. No cross-reaction was observed with control male plasma. A competitive enzyme-linked immunosorbent assay (ELISA) was developed using a-Vg and HA I. The detection limit of the assay was 6.25 ng/ml, and the intra- and inter-assay variations were 4.48 and 7.57%, respectively. Displacement curves parallel to the standard (HA I) were obtained with plasma samples from vitellogenic female and E2-treated male mrigal. The assay was validated by estimating Vg in plasma samples from adult female mrigal captured in the field throughout the year and compared with ovarian development. Annual profiles of plasma Vg and gonadotropin (GTH II: estimated by common carp GTH II ELISA) levels presented a good correlation with gonadosomatic index (GSI) and mean number of vitellogenic oocytes during different reproductive phases, i.e., preparatory (Feb–Apr), pre-spawning (May–Jun), spawning (Jul–Aug) and post-spawning (Sep–Jan).