University of Florida College of Veterinary Medicine, Aquatic Animal Health Program
Gainesville, FL, USA
Clinical methods previously attempted for pregnancy detection in manatees have been difficult to interpret,2 causing field biologists to rely upon visual indications of pregnancy that appear at approximately seven months gestation. These include distension of the abdomen and swelling of the vulva.1
Commercially available kits for serum progesterone (P4) measurement in domestic species have lower limits of 2.0 ng/ml (Endocrine Technology, Inc., Newark, CA) that make them too insensitive for use in manatees, where pregnancy is characterized by P4 values near 1.0 ng/ml. A high sensitivity serum P4 assay (IMMULITE 1000®, Diagnostic Product Corporation, Los Angeles, CA), with a lower limit of detection equal to 0.03 ng/ml is being validated at UFCVM as a tool for pregnancy diagnosis in the manatee.
Precision of our assay has been tested by determining inter-assay (<20%) and intra-assay (<15%) coefficients of variation, where variation less than 25% is accepted for immunoassays. Accuracy was determined and is also within acceptable limits defined by the Clinical Laboratory Improvement Amendment (2003). Methods comparison was completed by testing samples for P4 in an endocrine laboratory with a P4 assay validated for use in elephants. ROC Analyses based upon 38 non-pregnant females and 11 pregnant females indicated that once P4 values reach 0.5 ng/ml, the test approaches 100% certainty that a female manatee is pregnant. Conversely, when P4 reaches 0.03ng/ml, the test approaches 100% certainty that a female is not pregnant.
This assay, in final stages of validation, will help diagnose pregnancy during the first six months of gestation, before physical indicators appear, as well as provide additional confirmation of pregnancy in gestational months six through twelve. This information is invaluable for monitoring reproductive health of free-ranging and captive manatees, and will be necessary if assisted reproductive techniques are considered for sirenians.
Many thanks to Bob Bonde, Benjamin Morales, Greg Bossart, and FWRI staff for providing samples, and to Melanie Pate and Jim Burrow for assistance in the laboratory. Thanks also to Dennis Schmitt from the University of Missouri for running samples for our methods comparison experiment.
1. Walsh MT, GD Bossart. 1999. Manatee Medicine. In: Fowler, M.E., and R.E. Miller (eds.). Zoo and Wildlife Medicine, 4th ed. W.B. Saunders Co., Philadelphia, Pennsylvania. Pp.507-516.
2. Walsh M. Sea World of Florida. 14 February 2006. Personal communication.