NADP and NADPH was obtained from HiMedia Laboratories Pvt. Ltd, Mumbai, India. Reference standards for four Pregnen 20 ol three one and P4 have been obtained from Sigma Aldrich, Bangalore, India. Oligo dT and oligonucleotide primers have been synthesized by Sigma Genosys, Bangalore, India. The high performance liquid chromatography grade acetonitrile was obtained from Qualigens, Mumbai, India. All other reagents have been purchased from Sigma Aldrich, Bangalore, India or sourced via neighborhood suppliers. Animals, experimental protocol, blood and CL collection schedule Experiments in buffalo cows Non lactating adult buffalo cows aged 5 6 years having a identified history of regular cyclicity have been recruited for the study. Tissues including spleen, brain, skeletal muscle, kidney, mammary gland, lung, heart, liver, myometrium and CL have been collected to analyse the expression of 20 HSD across various tissues.
The day of onset of estrus was designated as day 0 of estrous cycle. To confirm the presence of functional CL, blood samples have been collected on days three to 7 from the cycle for monitoring circulating P4 concentration. Within this experiment, Juramate was administered 500 ug i. m, on day 11 of estrous cycle extra resources and CL was collected instantly before, 3, 6, 18 and 36 and 60 h post PGF2 injection. Blood samples were collected right away ahead of and at different time intervals post PGF2 injection for determining serum P4 levels. Ovaries containing CL were collected post slaughter and washed in sterile ice cold PBS and transferred into Dulbeccos Modified Eagles Medium supplemented with penicillin and streptomycin and transported towards the laboratory on ice inside 30 min of collection.
Beneath sterile circumstances, CL was extirpated, CCI-779 cut into eight to twelve pieces, transferred to labelled cryovials, snap frozen in liquid nitrogen and stored at ?70 C until analysis. Experiment in rats Impact of PGF2 remedy on luteal function in rats It really is well documented that PGF2 remedy increases 20 HSD expression in the CL and circulating 20 OHP in pseudo pregnant rats. We utilized pseudo pregnant rat model technique to serve as reference for PGF2 studies in buffalo cows. 3 month old adult female rats have been housed in a controlled environment and kept beneath a photoperiod of 12 h light and 12 h of darkness cycle with ad libitum access to meals and water. Pseudo pregnancy was induced in female rats by cohabitation with vasectomised male rats on the afternoon of proes trus.
Following cohabitation, female rats have been examined for the presence of vaginal plug and or subjected to screening of vaginal smears daily for the extension of your diestrus period. The presence of vaginal plug and or upon confirmation of day 1 of continuous diestrus following cohabitation with vasectomised male rats was designated as day 1 of pseudo pregnancy.