Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation

Abstract

In this study, 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 evaluated 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). Flow cytometry was also used to assess viability (percentages of live and dead cells) of boar sperm after staining with SYBR-14 and 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. The mean percentages of acrosome-intact and acrosome-reacted cells were 88.4 and 6.8 or 0.8 and 96.5 in semen treated with 0 or 100 microg/ml lysophosphatidylchloine (LPC), respectively (P < 0.001). Most spermatozoa were also in the AR CTC-stained category after treatment with LPC compared with a small proportion in the controls. 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 or 5 degrees C) and freezing reduced the 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 15 degrees C (56.1, 32.6, 11.5% F-, B-, and AR-pattern spermatozoa, respectively).