Development of a direct (non-extracted) enzyme immunoassay for measurement of serum progesterone in mares

Abstract

s / Journal of Equine Veterinary Science 33 (2013) 321-399 371 reproductive cyclicity in obese broodmares maintained on pasture. Eighteen healthy, light horse mares were matched by age and assigned to obese control (OBC; n1⁄45, mean BCS1⁄47.4 0.3), obese supplemented with 5g/d resveratrol (OBR; n1⁄47, mean BCS1⁄47.4 0.2) or non-obese control (NOC; n1⁄46, mean BCS1⁄45.4 0.1) treatments. Control horses received the resveratrol carrier placebo andNOChorseswere managed to maintain constant BCS throughout the trial. Across three consecutive estrous cycles, morphometric measurements were collected biweekly and follicular dynamics were evaluated via transrectal ultrasonography every other day to determine estrous cycle and follicular phase length, follicle size and number, and time of ovulation. Frequently-sampled intravenous glucose tolerance tests were conducted during luteal phase prior to supplementation and after third ovulation. Insulin and glucose kinetics were analyzed via minimal model. For all parameters, repeated measures (parametric or nonparametric, as warranted) techniques were used to determine treatment effect. Resveratrol supplementation had no discernible effect on reproductive parameters (P>0.05). Regardless of treatment (OBC or OBR), obese mares had more (7 vs. 0) hemorrhagic anovulatory follicles and tended (P1⁄40.07) to have longer inter-ovulatory intervals than NOC. Neither resveratrol treatment nor time on study influenced BCS, or bodyweight, rump fat thickness or neck circumference (raw data or data adjusted for animal size). Insulin sensitivity and disposition index were neither affected by resveratrol supplementation nor initial BCS, although as a whole, horses became more insulin resistant over time (P<0.05). NOC horses had lower (P<0.06) acute insulin response to glucose relative to OBC or OBR, but no difference (P1⁄40.19)was detected between obese groups. Although resveratrol supplementation did not elicit detectable responses in this study, promising results in other species warrant further investigation of the compound in horses, including exploration of bioavailability and possible effects at the tissue or cellular levels. Development of a direct (non-extracted) enzyme immunoassay for measurement of serum progesterone in mares R.M. Brooks , D.J. Denniston , J.E. Bruemmer , T.M. Nett , and P.M. McCue 3 Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523 Progesterone (P4) isa steroidhormoneproducedby theovary and placenta of the mare. Progesterone is required for the maintenance of pregnancyand anassessmentof endogenous concentration is useful in many diagnostic applications related to equine breeding management. The overall objectiveof thisstudywastodevelopandvalidateadirectenzymelinked immunosorbent assay (ELISA) for themeasurement of P4 in themare. The specific aimwas to develop a quantitative and sensitive progesterone assay that could be used on nonextracted equine serum or plasma. Significant events in the successful development of the ELISA included the use of purified anti-progesterone antibody, heterologous combination of antibody and conjugate, and methodology to avoid organic solvent extraction. It was determined that by lowering the volume of serumused for analysis and lowering the pH of the serum, a non-extracted assay could be accomplished. The overall correlation between ELISA of nonextracted serum and radioimmunoassay (RIA) of extracted serumwas high (r1⁄4 0.92) as seen in Figure 1.