Progesterone kit

Serum and plasma testosterone levels were determined following an enzyme immunoassay method validated for use with dolphins by Kita et al. [21 ]. Assay sensitivity for testosterone was 0.1 ng/ml. Levels of serum and plasma progesterone were determined using a commercial enzyme immunoassay kit manufactured by Neogen
Corporation (low-levels: Progesterone Ultra Kit #402410, 0.1–2 ng/ml; high-levels: Progesterone Kit #402310, 0.4–40 ng/ml) following manufacturer’s instructions.

The levels of plasma or serum progesterone were measured using commercially available enzyme immunoassay kits after diluting the samples twice (Progesterone Ultra Kit, #402410, standard
range: 0.1–2 ng/ml, Neogen; Lansing, MI, U.S.A.). The samples with high progesterone levels (>4 ng/ml) were reanalyzed
using an enzyme immunoassay kit with a higher standard range (Progesterone Kit, #402310, standard range: 0.4–40 ng/ml, Neogen). The low progesterone levels
in the samples obtained from F3 from November 2011 to the end of the study period were determined using a fluorescence immunoassay method with a fully-automated microfluidic immunoassay
system (Mini Vidas SV-5010, SYSMEX bioMerieux; Tokyo, Japan), because the additional Progesterone Ultra Kits were not available in Japan at the time of our study. All procedures were
performed according to the manufacturer’s instructions. The intra- and inter-assay coefficients of variation were less than 10% for all three methods. The WBC counts were analyzed with an
automated hematology analyzer (KX-21NV, Sysmex; Kobe, Japan).