Development and Validation of a Sensitive Radioimmunoassay for Progesterone Estimation in Unextracted Mithun (Bos frontalis) Plasma

Abstract

The objective of the study was to develop and validate a simple, reliable, and highly sensitive radioimmunoassay (RIA) for progesterone determination in mithun (Bos frontalis) plasma. The RIA was carried out in 20 µL unextracted mithun plasma. The progesterone standards ranging from 2 to 500 pg/20 µL/tube were prepared in charcoal‐treated hormone‐free plasma. The sensitivity of the RIA procedure was 2 pg progesterone/20 µL/tube, which corresponds to 0.1 ng/mL; the 50 percent relative binding sensitivity was seen at 32 pg/20 µL/tube. Plasma volumes for the RIA, viz. 10 and 20 µL, did not influence the shape of standard curve, even though a slight drop in the counts was seen with higher plasma volumes. For the biological validation of the assay, three cyclic, three in early pregnancy, and two in late pregnancy mithuns were used. Blood samples collected at weekly intervals for 42 days, from all animals, were assayed for plasma progesterone. The peak level of progesterone was registered at day 14 (day 21 of sampling) of the estrous cycle and the lowest at estrus; the progesterone concentrations increased and decreased gradually as sampling time advanced, in early and late pregnancy, respectively, which confirm the biological validation of the RIA. The RIA avoids the troublesome and laborious plasma extraction procedures. In conclusion, the RIA developed for progesterone determination in mithun blood plasma is sufficiently reliable, simple, and sensitive enough to estimate progesterone in all physiological variations in mithun.

Keywords:

• Mithun‐endocrinology
• Progesterone
• RIA
• Validation
• Plasma
• Semi‐wild

Acknowledgments

The authors wish to thank Dr. Vijay Paul, Research Associate, National Bureau of Animal Genetic Resources, Karnal, India and Dr. Kajal Shankar Roy, PhD Scholar, Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal, India for technical assistance during the study. The authors also wish to thank the Director, National Dairy Research Institute, Karnal‐132 001 (Haryana), India for providing part of the facilities for the present work.