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Title: CHLORTETRACYCLINE ANALYSIS OF BOAR SPERMATOZOA AFTER INCUBATION, FLOW CYTOMETRIC SORTING, COOLING, OR CRYOPRESERVATION

Author
item MAXWELL, W.M.C. - UNIVERSITY OF SYDNEY
item Johnson, Lawrence

Submitted to: Journal of Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/20/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Flow cytometric sorting of X and Y chromosome bearing sperm to use for sex preselection is available on a limited scale at the practical level. This study was designed to find ways of improving the viability over time of sperm that have been processed with the Beltsville Sperm Sexing Technology. It was found that sperm passing through the cell sorter are capacitated prematurely. The need for a method to preserve sorted sperm after sorting requires adjustment. This information will be used by scientists to improve the Beltsville Sperm Sexing Technology.

Technical Abstract: The effects of staining procedure with chlortetracycline (CTC) and method of analysis of boar spermatozoa after staining were examined. The hypothesis that incubation, flow cytometric sorting, cooling, and cryopreservation cause changes to boar sperm membranes which resemble capacitation and the acrosome reaction was also tested. Membrane status was sevaluated by flow cytometry and by fluorescence microscopy after staining with CTC, and acrosome integrity was checked by flow cytometry after staining with FITC-pisum sativum agglutenin and propidium iodide (PI). Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of spermatozoa allocated to the F(uncapacitated), B(capacitated), or AR (acrosome-reacted) CTC fluorescent staining categories. Using flow cytometry to examine sperm suspensions stained with CTC, a gated population of spermatozoa with low fluorescence (population 1) comprised predominantly F-pattern cells (F- pattern: population 1 vs. population 2, 80.5 vs. 14.4%; P < 0.001), whereas population 2 (high fluorescence) comprised mainly B-pattern cells (B- pattern: population 1 vs. population 2, 8.5 vs. 62.3%; P < 0.001). Incubation (38 degrees C, 4 hr), flow sorting, cooling (to 15 degrees C) and freezing reduced with proportion of F-pattern and live spermatozoa, and increased the proportion of B-, AR-pattern, and dead spermatozoa, in comparison with fresh semen. There were more membrane changes in spermatozoa cooled to 5 degrees C (30.4, 48.5, 21.1%) than in those cooled to 15o C (56.1,32.6,11.5% F-,B-, and AR-pattern spermatozoa, respectively).