Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa.
OBJECTIVE
To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa.


METHODS
Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions.


METHODS
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 flash-frozen 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% paraformaldehyde (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
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 from these species.
Brum
Andrea M
AM
Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Thomas
Alysia D
AD
Sabeur
Khalida
K
Ball
Barry A
BA
D000177:Acrosome / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D004285:Dogs; D006736:Horses; D007202:Indicators and Reagents / Q000032:analysis* / Q000737:chemistry; D016015:Logistic Models; D008297:Male; D012394:Rosaniline Dyes / Q000032:analysis* / Q000737:chemistry; D012662:Semen Preservation
D007202:Indicators and Reagents; D012394:Rosaniline Dyes; C048139:Coomassie blue