Determination of plasma kisspeptin concentrations during reproductive cycle and different phases of pregnancy in crossbred cows using bovine specific enzyme immunoassay.

Kisspeptin, a decapeptide and potent secretagogue of GnRH has been emerged recently as a master player in the regulation of reproduction in animals. Determination of kisspeptin in peripheral circulation is, therefore, very important for studying the control of its secretion and its role on reproduction in bovine species, the information on which is not available during any physiological state in this species, may probably be due to non-availability of simple assay procedure to measure the hormone. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for kisspeptin determination in bovine plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to kisspeptin and used to bridge between streptavidin-peroxidase and the immobilized kisspeptin antiserum in the competitive assay. The EIA was conducted directly in 100 μl of unknown bovine plasma. Kisspeptin standards ranging from 0.01 to 25.6 ng/100 μl/well were prepared in hormone-free plasma. The lowest detection limit was 0.1 ng/ml plasma. Plasma volumes for the EIA, viz., 50, 100 and 200 μl did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous bovine kisspeptin with kisspeptin standard used. It showed good parallelism with the kisspeptin standard curve. For the biological validation of the assay, plasma kisspeptin was measured in blood samples collected from six non-lactating cyclic cows during entire estrous cycle and from 18 pregnant cows during different stages of pregnancy. The mean plasma kisspeptin concentration during different days of the estrous cycle was different (P<0.001). Three peaks of kisspeptin were recorded, one on a day before appearance of preovulatory LH surge, second at day 6 and third one at day 18 of the estrous cycle. Plasma kisspeptin concentrations increased (P<0.001) from first through last trimester of pregnancy. Kisspeptin concentrations were also measured in different follicular, luteal and placental tissues. Follicular and placental kisspeptin levels increased (P<0.01) during follicular development and with the advancement of pregnancy, respectively. On the other hand, luteal concentrations of kisspeptin decreased (P<0.01) with its developmental process. In conclusion, a simple, sufficiently sensitive and direct EIA procedure has been developed for the first time to determine plasma kisspeptin levels in bovine. A wide range of kisspeptin concentrations can be detected during different physiological stages in bovine using this kisspeptin-EIA procedure.
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