Use of a monoclonal antibody to evaluate integrity of the plasma membrane of stallion sperm.

This research article discusses a method of using a particular monoclonal antibody to review the integrity of the plasma membrane in stallion sperm. The approach utilizes transmission electron microscopy and an indirect immunofluorescent process and proves that it is possible and reliable to determine sperm health through this method.

Objective

The objective of the research was to develop and substantiate a procedure using a monoclonal antibody (F79.3E2; class IgG1 kappa) to assess the integrity of the plasma-acrosomal membranes of stallion sperm.

Methodology

• The research used transmission electron microscopy to prove that the F79.3E2 antibody was specifically localized to an antigen in the acrosomal ground substance of stallion sperm.
• The basis of the study was that the primary antibody would be “shielded” from its acrosomal antigen by an intact plasma membrane. Alternatively, if the plasma-acrosomal membranes were damaged, the sperm would exhibit green fluorescent due to the antibody.
• To make sure all sperm were visible, a lipophilic counterstain producing red fluorescence was used.
• Sperm in fresh-extended or frozen-thawed semen were incubated with the hybridoma supernatant containing the monoclonal antibody for 30 minutes at 37 degrees Celsius.
• They then added a second antibody (rabbit anti-mouse IgG-FITC) for another 30 minutes at the same temperature.
• After the unbound antibody was removed by dilution and centrifugation, the sperm were resuspended in phosphate-buffered saline mixed with a counterstain called Evan’s Blue.

Results

• All sperm fluoresced bright red despite the condition of cell membranes. If the plasma-acrosomal membranes were damaged, the green fluorescence from the antibody was dominant in the rostral portion (front part).
• The percentage of sperm with intact plasma-acrosomal membranes could be easily determined by counting fluorescent and nonfluorescent “acrosomes”.
• The correlation score was 0.91 between the percentage of damaged sperm in a mixture and the percentage of sperm with a fluorescent acrosome, indicating a high accuracy rate of this process for determining sperm damage.
• Both intra-assay and inter-assay coefficients of variability were less than 6%, providing evidence towards the stability and reliability of this evaluation method.