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Title: MEMBRANE STATUS OF BOAR SPERMATOZOA AFTER COOLING OR CRYOPRESERVATION

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

Submitted to: Theriogenology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/14/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Improving the Beltsville Sperm Sexing Technology is critical to its greater utilization in the livestock industry. This manuscript details the effects of sorting, cooling and cryopreservation on the membrane integrity of boar sperm. Earlier data had suggested that sperm subjected to such treatment as flow cytometric sorting and cryopreservation individually showed greater ramounts of capacitation, thus enhancing their readiness for fertilization These experiments quantified those changes through the use of fluorescent staining to detect the changes in the acrosome. Holding sperm in their original plasma was helpful to maintaining the viability of the sperm after sorting, as well as after cooling, freezing and thawing and flow cytometric sorting. These results are useful to scientists for developing improved protocols for handling sorted sperm, in order to provide for practical application in various species.

Technical Abstract: Capacitation status was evaluated by flow cytometry and by fluorescence microscopy after staining with chlortetracyline (CTC), and acrosome integrity was determined by flow cytometry after staining with FITC-pisum sativum agglutenin and PI. Staining of spermatozoa with CTC alone and in combination with PI and/or Hoechst 33342 had no effect on the proportion of fspermatozoa 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 ug/ml lysophophatidylchloine (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 suspension 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 than to 15 degrees C. The proportion of live spermatozoa decreased during processing for cryopreservation and cooling to 5 degrees C, but was not affected by freezing and thawing if held at 15 degrees C for 3.5 hr during cooling.