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Abstract
Erin Reynolds

Fellow – Erin Reynolds

Mentor – Katrin Hinrichs

Project – Verification of CatSper Protein in Equine Sperm

Objective - In vitro fertilization (IVF) is not currently effective in horses. The failure of IVF is thought to be related to inability of the sperm to gain hyperactivated motility. In other species, the CatSper channel is responsible for the calcium influx that triggers sperm hyperactivation. This study was conducted to verify the presence of the CatSper channel in equine sperm.

Animals or Sample Population – Semen was collected from a healthy, fertile stallion.

Procedure – Two separate experiments were performed. In Experiment 1, immunocytochemistry was performed using a rabbit polyclonal antibody against human CatSper1 (Antibody A). Because no blocking antigen was available for Antibody A, immunocytochemistry was attempted with a second antibody raised against a smaller human CatSper1 segment (Antibody B), as well as with two antibodies against human CatSper2. In Experiment 2, an immunoprecipitation was performed using Antibody A conjugated to Dynabeads® followed by one-dimensional gel electrophoresis. Mass spectrometry was performed on the bands.

Results – Seven replicates of the immunofluorescent staining were performed. All trials with Antibody A demonstrated clear staining of the principal piece of the sperm. Absence of the primary antibody resulted in no visible staining. Neither Antibody B nor the two antibodies against CatSper2 stained the horse sperm. Two of the immunoprecipitation gel blots showed distinct bands, both at about 60,000 kDa.

Conclusion and Clinical Relevance – Identification of the CatSper1 protein verifies the presence of the CatSper channel in equine sperm. This channel requires further study to explain the lack of hyperactivation during in vitro fertilization.