# Application of Staining Methods to Compare Chromatin Condensation in Fresh and Freeze‐Thawed Dog Semen

## TL;DR

This study compares different staining methods for analyzing chromatin condensation in dog semen, finding that toluidine blue works well for light microscopy and CMA3 is best for fluorescence microscopy.

## Contribution

The study provides a direct comparison of multiple staining techniques using identical canine semen samples, identifying optimal methods for different microscopy approaches.

## Key findings

- Aniline blue staining showed significant differences compared to toluidine blue, AcO, and CMA3.
- CMA3 is recommended for fluorescence microscopy due to its simplicity, rapidity, and cost-effectiveness.
- Correlations between staining methods varied, with AcO showing strong correlations with aniline blue and CMA3 in both fresh and frozen samples.

## Abstract

Various staining techniques have been used for canine sperm analysis, but direct comparisons using identical semen samples are lacking. This study aimed to assess the efficiency, time requirements and cost‐effectiveness of different staining techniques: aniline blue, toluidine blue, acridine orange (AcO), chromomycin A3 (CMA3) and terminal deoxynucleotidyl transferase d‐UTP nick‐end labeling (TUNEL). Forty semen samples (20 fresh and 20 frozen–thawed) were used to assess chromatin condensation. Significant differences (p < 0.01) were found using two‐factor repeated measures variance analysis. Aniline blue staining differed significantly (p < 0.01) from toluidine blue, AcO and CMA3 staining. A significant difference (p < 0.05) was observed between AcO and TUNEL staining for fresh sperm, with no significant differences between TUNEL and other methods. Correlations in fresh sperm samples showed r = 0.567 between AcO and aniline blue, r = 0.645 between AcO and CMA3, and correlations of 0.455 and 0.557 for aniline blue – toluidine blue and aniline blue – TUNEL, respectively. For frozen samples, significant differences were found between aniline blue and toluidine blue and AcO tests (p < 0.05), and between CMA3 and TUNEL staining (p < 0.01). Correlations in frozen samples showed r = 0.582 between AcO and aniline blue, r = 0.752 between AcO and CMA3, r = 0.698 between toluidine blue and aniline blue, and r = 0.536 between CMA3 and aniline blue. A minimal association was found between standard semen analysis and chromatin analysis. In conclusion, toluidine blue is effective for light microscopy staining, while CMA3 is recommended for fluorescence microscopy due to its simplicity, rapidity and cost‐effectiveness.