Abstract

Sixteen serum samples from Tursiops truncatus with progesterone concentrations ranging from 0.2 to 45.18 ng/ml as determined by radioimmunoassay also were assayed using a semi-quantitative enzyme immunoassay test kit. Equivalent results were obtained suggesting that this EIA kit may provide a simple, cost-effective alternative to RIA for estimating progesterone status in the Atlantic bottle-nosed dolphin. Keywords: Bottle-nosed dolphin, progesterone, enzyme immunoassay, Tursiops truncatus, pregnancy diagnosis.

Introduction

The steroid hormone, progesterone (P), is critical to the initiation and maintenance of pregnancy in mammals. Determining P concentrations indicates the presence or absence of a functioning corpus luteum, information useful in confirming reproductive status.^1,3,7,9 Traditionally, P concentrations have been determined by radioimmunoassay (RIA), a procedure requiring the use of radioactive isoto pes and laborious laboratory techniques. The advent of enzyme immune assay (EIA) test kits^1,2,3,4,7,9 now permit easy, on-site determination of P concentrations in horse and cattle serum or plasma and bovine milk. ^1,3,7,9 Results are known within an hour at a fraction of the cost incurred by conventional RIA procedures.

Sequential determination of P concentrations is an important tool in diagnosing pregnancy in the bottle-nosed dolphin^5,6 Although definitive data on the reproductive cycle still are unavailable, dolphins are considered to be seasonally polyestrous,^5,6,8 exhibiting zero to three estrous cycles per year^5,6 with an average length of 23-30 days.⁵ single serum sample with a P concentration greater than 3 ng/ml is indicative of ovulation or pregnancy.⁵ A presumptive diagnosis of pregnancy can be made when an animal with a history of exposure to a male has elevated P concentrations for longer than six to eight weeks. Four elevated samples drawn at two week intervals are recommended for a definitive diagnosis of pregnancy. (V. L. Kirby, pers.com.)^5,7

This preliminary study was conducted to determine if an EIA test kit designed for assaying P concentrations in equine serum or plasma could be used to estimate P status in the bottlenose dolphin.

Materials and Methods

Thirteen samples from the Dolphin Research Center (DRC) were banked sera that had been stored at -20°C for various lengths of time ranging from 1 month to 5 years. This serum had been collected from 8 adult female bottle-nosed dolphins located at DRC. It had been assayed for P concentrations by RIA at the time of collection. Five samples had been collected from non-pregnant animals, 7 samples from pregnant animals, and 1 from an animal tentatively diagnosed as having aborted or had cystic ovaries. Three blood samples from non-pregnant animals were collected during routine physical exams and assayed by EIA and RIA before freezing.

Samples were analyzed by RIA at Roche Laboratories, (Roche Biomedical Laboratories, 1447 York Court, Burlington, North Carolina 27215, U.S.A.) using a monoclonal antibody. Fresh and frozen samples were assayed by EIA under field conditions at DRC. The monoclonal antibody based enzyme immunoassay kit (Cite Semi-Quant^TM, IDEXX, 100 Fore Street, Portland, Maine 04101, U.S.A.) used was designed as a semi-quantitative test for P in equine serum or plasma. Each assay device contained a fiber membrane supported by a porous disk with a cellulose absorbent in the base. The fiber membrane had three spots: two contained calibrated levels of monoclonal antibodies to progesterone and one was a positive control. P concentrations were categorized as less than 1 ng/ml, 1 to 4 ng/ml and greater than 4 ng/ml. Results were determined by changes in the pattern of color development among the three spots on the filter. Because this was a competitive EIA, color development was inversely proportional to P concentrations.

The procedure for the enzyme immunoassay was followed according to the manufacturer's guidelines. The reagent tray and assay devices were stored at 36-45°F and allowed to equilibrate to room temperature (72°F) for at least 45 min. before use. Thawed serum samples were centrifuged for 5 min. before testing. The assay device was prepared by first adding the manufacturer's wash solution (0.75 ml), then serum or plasma (0.75 ml). After a 10 min. incubation, the device was washed (2.0 ml) before adding enzyme conjugated P (0.2 Ml). After exactly 1 min., the device was washed (2.0 ml), and an enzyme substrate/chromagen solution (0.2 ml) was added to begin color development. A stop solution was added after 1 min. The reaction was read both during color development and after the reaction was stopped.

Results and Discussion

Concentrations of P as determined by RIA and EIA are depicted in Table 1. The EIA results yielded five samples in the < 1 ng/ml range, three in the 1-4 ng/ml range and eight in the >4 ng/ml range. Although the number of samples assayed was small, the EIA and RIA results were in correspondence on all occasions.

This EIA appeared to provide reliable results on P status when the manufacturer's guidelines were followed exactly. Although the procedure is simple, our experience indicates that mistakes can occur when the reagents and serum are not in the correct temperature range (68-75°F). If the control spot on a device is lighter than the norm for that kit, a handling error may have occurred, and the assay should be repeated. (R. Lord, pers. comm.) Centrifugation of thawed samples is recommended to prevent obstruction of the membrane by sediment. Although this EIA does not provide an absolute hormone value, it does provide sufficient information potentially suitable for predicting ovulation or pregnancy.

Use of P concentrations to diagnose pregnancy in the bottle-nosed dolphin has several advantages over other methods. An obstetrical doppler can be used to detect a fetal heartbeat but only in the second trimester.^10,11 Ultrasound is effective at any stage of pregnancy,^11,12 but the expensive electronic equipment is sensitive to water and salt air and may require stressing the animal by removing it from the water. However, blood can be collected for P determination at any time during the pregnancy. Many animals are trained for fluke presentation so the procedure can be performed in the water with minimal labor.¹⁰ The availability of a progesterone EIA kit would further simplify pregnancy evaluation in the bottle-nosed dolphin.

Acknowledgements: The author thanks Jayne Rodriguez and Lynn Calero, Dolphin Research Center, Grassy Key, Florida for the use of the Center's serum bank. The author also thanks Dr. Larry Chaney and Rob Lord from Cite Technical Services, IDEXX, Portland, Maine, for their technical assistance and for supplying the EIA kits.

Table 1. Comparison of results on serum progesterone assays using RIA and EIA

References

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