Methodological improvement of fluorescein isothiocyanate peanut agglutinin (FITC-PNA) acrosomal integrity staining for frozen-thawed Japanese Black bull spermatozoa
| Autor: | Koushi Mukoujima, Hiromichi Adachi, Quzi Sharmin Akter, Tetsuma Murase, Reza Rajabi-Toustani, Yoichiro Hoshino, Essam A. Almadaly |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
Peanut agglutinin
Male 040301 veterinary sciences bull spermatozoa Fluorescein isothiocyanate-peanut agglutinin 0403 veterinary science Andrology 03 medical and health sciences chemistry.chemical_compound Peanut Agglutinin Animals acrosomal integrity Fluorescein Paraformaldehyde 030304 developmental biology Cryopreservation 0303 health sciences FITC-PNA staining General Veterinary biology Full Paper Staining and Labeling Chemistry 04 agricultural and veterinary sciences Fluoresceins Sperm Spermatozoa Staining biology.protein Cattle Theriogenology Acrosome Semen Preservation |
| Zdroj: | The Journal of Veterinary Medical Science |
| ISSN: | 1347-7439 0916-7250 |
| Popis: | This study aimed to improve the staining of frozen-thawed Japanese Black bull sperm acrosomes with fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Spermatozoa were washed, fixed with 1-3% paraformaldehyde (PFA) in suspension for 10, 20, and 30 min, permeabilized with 0-2% Triton X-100 for 5 min, stained with FITC-PNA, and mounted with different antifade agents (0.22 M 1,4-diazabicyclo [2,2,2] octane (DABCO), SlowFade®, and ProLong®) in suspension (In-suspension) or on a smear (On-smear). The spermatozoa were categorized into seven pattern types either immediately or after storage for 24 hr. Experiment 1 showed that 1) the In-suspension method was better than the On-smear method; 2) if spermatozoa were stained using the In-suspension method and examined immediately, the best antifade agent was SlowFade®; 3) if samples were to be stored after staining using the On-smear method, DABCO should be avoided; 4) if spermatozoa were stained using the In-suspension method, storage of the stained samples was not recommended; and 5) if samples were to be stored after staining using the In-suspension method, ProLong® might be the best antifade agent. The results of experiment 2 showed that the concentration of Triton X-100 could be reduced to 0.1 from 1%. The results of experiment 3 showed that the paraformaldehyde concentration used for a 30 min fixation could be reduced from 3 to 2%. It is expected that the improved staining protocol will be useful to determine bull sperm acrosomal integrity. |
| Databáze: | OpenAIRE |
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