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Lindsey Brehm

Fellow – Lindsey Brehm

Mentor – Dickson Varner, Charles Love

Project – Comparison of TUNEL and SCSA assays for measurement of stallion sperm DNA/chromatin quality before and after centrifugation through a silica-particle solution

Objective – To compare the effectiveness of these techniques for assessing sperm chromatin quality

Background – DNA/chromatin damage has been examined in conjunction with more conventional methods of determining semen quality, i.e., motility and morphology, in an effort to provide a more accurate determination of sperm functionality. Sperm DNA/chromatin damage has been studied extensively due to the negative effects that the damage can have on fertilization rate and embryo development and viability. Sperm DNA damage has already been associated with reduced natural conception, intrauterine insemination, and in vitro fertilization outcomes. The Sperm Chromatin Structure Assay (SCSA) is considered the standard for identifying DNA damage. The Tdt-mediated-dUTP nick end labeling (TUNEL) assay has recently been shown to be another effective method for evaluating sperm chromatin. Work done previously in this laboratory has shown that centrifuging sperm through a silica-particle solution can be used to select for sperm with normal morphology and good chromatin quality. The discontinuous gradient centrifugation separates sperm based on isopycnic points, leaving primarily poor quality sperm at the top and allowing higher quality sperm to accumulate at the bottom of the centrifuge tube.

Procedure – A portion of stallion ejaculates is immediately frozen, while another is centrifuged by discontinuous gradient method, using a silica-particle solution. Both centrifugation portions are then evaluated using the SCSA and the TUNEL protocol validated previously in this laboratory.

Results – The results have shown that the TUNEL assay does detect sperm DNA/chromatin damage as effectively as the SCSA and that centrifugation through a silica-particle solution separates sperm based on chromatin quality.

Conclusions – Either technique has value as an adjunctive assay to incorporate in a breeding soundness examination of stallions.