Application of Enzyme-Linked Fluorescence Assay (ELFA) to Obtain In Vivo Matured Dog Oocytes through the Assessment of Progesterone Level

Abstract

Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D − 6 to D + 1) in both systems were compared. The ELFA range of 5.0–12.0 ng/mL was derived from the RIA standard range of 4.0–8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.

Keywords

• enzyme-linked fluorescence assay
• serum progesterone
• somatic cell cloning
• somatic cell nuclear transfer
• dog