article · Journal of Veterinary Medical Science
This study aimed to improve the staining method for frozen-thawed Japanese Black bull sperm acrosomes using fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Researchers optimised parameters including paraformaldehyde concentration, Triton X-100 concentration, and antifade agents. They compared in-suspension versus on-smear methods and assessed immediate versus stored sample examination. Key findings indicated that the in-suspension method was generally superior. For immediate examination, SlowFade® was the best antifade agent, while for stored in-suspension samples, ProLong® might be preferable. The study also found that Triton X-100 concentration could be reduced to 0.1% and paraformaldehyde to 2% for 30-minute fixation. The improved protocol is expected to aid in determining bull sperm acrosomal integrity.
Accurately assessing sperm quality is crucial for successful animal breeding programmes. This improved staining method provides a more reliable way to evaluate the acrosomal integrity of bull sperm, which is vital for fertilisation. This can lead to better selection of breeding animals and improved reproductive outcomes.
This research provides an improved, more reliable protocol for assessing bull sperm acrosomal integrity, which is a critical indicator of sperm quality. This could be applied in veterinary diagnostics, animal breeding centres, and artificial insemination programmes to enhance quality control and selection of viable semen. The improved protocol is an applied research outcome, offering a refined technique for immediate use in laboratory settings.
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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.
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DOI: 10.1292/jvms.18-0560
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