A Direct Time-resolved Fluorescent Immunoassay(TR-FIA) for Measuring Plasma Progesterone Concentration in the Sika Doe (Cervus nippon centralis).

Abstract

Abstract.A direct time-resolved fluorescent immunoassay (TR-FIA) system was developed and evaluated for measuring progesterone (P) concentrations in deer plasma. The minimum detectable level of this assay system was 0.25 ng/ml and ED 50 value of standard curve was 2.4 ng/ml. Intraand inter-assay coefficients of variation were 8.8% and 15.7%, respectively. The direct TR-FIA revealed that the plasma P concentrations in sika deer expressed a diurnal pattern depending on their reproductive status. Results in the present study indicate that the TR-FIA system, that is specifically designed for humans if successfully utilized with slight modification, can be utilized for direct assaying of P in deer plasma. The plasma P profile in the sika deer appears to be consistent to that of other temperate deer expressing seasonal breeding characteristics. . In Japanese mainland, the reproductive behavior of wild sika deer is manifested between October to December, with the delivery of newborn in early summer following 217 days of gestation [6]. Their reproductive organs sh o w c h a r a c ter is t ic c ha n g e s a c c o r d in g t o reproductive status and exert influence on the plasma hormone profiles in both genders [7,8]. In t h e r e i n d e e r , p l a s m a p r o g e s t e r o n e ( P ) concentration well reflects the luteal function and it has been a useful parameter for determining the repr oductive sta tus [9], h owever de ta iled information about plasma P concentrations in the sika deer is still limited.There are two major problems in analyzing P concentration in wild deer; one is the difficulty of serial sampling from animals and the other is a reliable and safe assay procedure. In reports published to date, plasma P analysis in the deer has been carried out by radioimmunoassay [10][11][12]. The handling of radioactive materials is strictly regulated by law and procedures have to be carried out in designated areas. Therefore a simple procedure for measuring plasma P concentration in the deer, that could be routinely carried out in a conventional laboratory would be of tremendous advantage.The time-resolved fluorescent immunoassay (TR-FIA) is one of the immunoassays employing

Annual Changes in Serum LH and Testosterone Concentrations in Male Sika Deer (Cervus nippon).

“…Sika deer are seasonal breeders that mate in the fall [3] and it is essential f o r e f f e c t i v e f a r m i n g t o u n d e r s t a n d t h e reproductive endocrinology of this species. Little is known about the reproductive endocrinology of sika deer except for the steroid hormones such as testosterone (T) in males [4][5][6] and progesterone in females [5,7]. Yamaji et al [8] described seasonal changes in the gonadotropic cells in the pituitary of wild sika deer.…”

A Direct Time-Resolved Fluoroimmunoassay (TR-FIA) for Measuring Plasma Estradiol-17.BETA. Concentrations in Cattle

“…It is based on time-resolved fluorometry instead of radiation counting, and is potentially more sensitive than radiation counting [2]. This type of assay has been used for plasma and fecal progesterone analyses in deer [15,16]. We did several preliminary trials according to the manufacturer's instructions to establish direct assaying for E 2 in bovine plasma.…”

“…The standard solution was prepared by adding E 2 (Sigma, St. Louis, MO, U.S.A.) to charcoal-treated plasma (collected from adult Friesian cows). The charcoal treated plasma was prepared as previously reported (15). The E 2 concentration of the charcoal-treated plasma was confirmed to be below the detectable level of TR-FIA (<1.25 pg/ml).…”

“…Progesterone assay: Plasma progesterone (P) concentrations were measured with the TR-FIA kit (DELFIA TM Progesterone Reagent, Wallac, Turku, Finland) as previously reported [15]. All standards and samples were assayed in duplicate.…”

“…At present, the reagents for TR-FIA are available for measuring E 2 , but it should be kept in mind that direct plasma immunoassays are sometimes affected by unknown factors present in the plasma. This has resulted in incorrect assay values when compared to the data of extraction assays [15]. The manufacturers of DELFIA TM Estradiol Reagents used in the present study recommend using 25 µl of plasma sample per assay.…”