Preliminary Studies in the Development of a Mare-side ELISA Test of Uterine Proteins as a Predictor of Breeding Soundness

Alexandre Daley and Regina Turner
University of Pennsylvania
and
Nancy Diehl, Alan Ealy, and Gary Killian
The Pennsylvania State University

Currently, evaluation of breeding soundness in mares involves obtaining an endometrial biopsy and evaluating it histologically. Evaluation of the histopathologic changes in the endometrium can be highly predictive of a mare’s fertility (Kenney, 1978; Kenney and Doig, 1986). However, obtaining and evaluating an endometrial biopsy requires a high degree of technical expertise. Additionally, it typically takes several days before the results of the test are reported back to the mare manager. The test also is relatively expensive to perform.

The long-term goal of this project is to create an inexpensive, mare-side test for breeding soundness that will complement the endometrial biopsy. The hypothesis is that differences in protein expression are present in endometrial biopsy samples from subfertile and infertile mares (Endometrial biopsy categories IIA, IIB and III) compared to fertile mares (Endometrial biopsy category I). If present, these differences in protein expression could be exploited to develop ELISA assays to aid in differentiating fertile from subfertile mares. The goal of our summer work is to obtain endometrial biopsy samples as well as uterine lavage samples from a group of cycling mares. Endometrial biopsy samples will be examined histologically and graded according to the Kenney system. Endometrial and lavage samples also will be sent to blinded collaborators at The Pennsylvania State University where protein will be extracted and protein extracts will be used for 2-dimensional protein gel electrophoresis. Attempts will be made to identify proteins that are differentially expressed in different samples. We then will attempt to correlate differentially expressed proteins with Kenney endometrial biopsy grades. If specific proteins are identified, effort will focus on testing the efficacy of using the identified proteins as markers for breeding soundness.

In the long run, we hope to be able to produce an enzyme-linked immunoassay that could be used mare-side as a rapid screening test for subfertility. In addition, these experiments will provide insight into the basic understanding of how changes in the endometrium affect reproductive performance. For two heat cycles, sixteen mares will be teased daily until heat commences. Palpation and ultrasonography will then be performed every other day until the first day of a detected 35 mm follicle. In the first cycle, a complete breeding soundness examination, including the collection of two endometrial biopsy samples, will be performed. One biopsy will be fixed and used for standard analysis of endometrial biopsy category. The other will be frozen for later protein extraction. In the second cycle, lavage fluid will be collected for later 2-D protein gel electrophoresis analysis.

References

Kenney RM 1978. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. J Am Vet Med Assoc 172:241-62.

Kenney RM, and PA Doig 1986. Equine endometrial biopsy. In Current Therapy in Theriogenology 2. DA Morrow (Ed) WB Saunders, Philadelphia. 723 - 729.

Alexandre Daley is a member of the Penn Vet Class of 2005.  She is working with Dr. Regina Turner for the summer as a research assistant.