TY - JOUR
T1 - Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa
AU - Brum, Andrea M.
AU - Thomas, Alysia D.
AU - Sabeur, Khalida
AU - Ball, Barry A.
PY - 2006/2
Y1 - 2006/2
N2 - Objective - To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. Sample population - Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. Procedure - Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flashfrozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. Results - Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformalclehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. Conclusions and clinical relevance - Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples f rom these species.
AB - Objective - To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. Sample population - Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. Procedure - Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flashfrozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. Results - Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformalclehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. Conclusions and clinical relevance - Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples f rom these species.
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U2 - 10.2460/ajvr.67.2.358
DO - 10.2460/ajvr.67.2.358
M3 - Article
C2 - 16454645
AN - SCOPUS:33344465356
SN - 0002-9645
VL - 67
SP - 358
EP - 362
JO - American Journal of Veterinary Research
JF - American Journal of Veterinary Research
IS - 2
ER -