Evaluation of different staining techniques for determination of membrane status in spermatozoa.
Proceedings of the New Zealand Society of Animal Production, Volume 57, pp 246-250, Jan 1997
Article class: Conference PresentationNew Zealand Society of Animal Production
AbstractChanges in the composition and integrity of spermatozoal membranes occur both during the normal capacitation and acrosomal reactions involved in the fertilization processes and as a result of different handling and storage procedures. Techniques to monitor these changes are essential to any study on methods of sperm preservation and such techniques should be both accurate, simple and rapid to apply. A series of tests were conducted to evaluate a range of staining techniques to measure the integrity of the sperm plasma membrane (permeable membrane indicates non-viable sperm) and to measure the status of the acrosomal membrane (indication of capacitation and acrosomal reaction). The possibility of a combination of stains to provide simultaneous answers to both parameters was also attempted. Semen both fresh and frozen from both rams and bulls was used. The stains evaluated were: Nigrosin-Eosin (NE), Nigrosin-Eosin- Giemsa (NEG), Hoechst 33258 vital stain as both wet and dry mounts, Carboxyfluorescein and Propidium Iodide (CF-PI), SYBR-14 and Propidium Iodide (SYBR-PI), Pisum sativum -fluorescein isothiocynate (PSA-FITC), Chlortetracycline (CTC) and a FITC- labelled monoclonal-antibody (Mab) to an acrosomal protein. The techniques were graded for their accuracy, ease of use, equipment requirements and also for repeatability or adaptability to spermatozoa in different media. No single or combination of techniques proved suitable for both plasma and acrosomal membranes and most techniques varied in their suitability for different semen preparations.
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