# Methods Used to Assess Bull Sperm Chromatin Integrity and Its Correlation with In Vitro Embryo Production Efficiency

## TL;DR

This study compares methods to assess bull sperm chromatin integrity and finds that acridine orange staining and TEM are most effective for predicting in vitro embryo production success.

## Contribution

The study identifies AO staining and TEM as low-cost, effective methods for evaluating bull sperm chromatin integrity in field and lab settings.

## Key findings

- AO staining and TEM showed strong correlations with in vitro embryo production efficiency.
- Chromatin alterations detected by AO and TEM were most effective in predicting cleavage and embryonic development rates.
- Alternative methods like TB and FR were less effective compared to AO and TEM.

## Abstract

Sperm chromatin alterations are often a cause of subfertility in breeding bulls. Despite the significance of this pathology in bovine reproduction, it is frequently overlooked by veterinarians, as the standard method for evaluating chromatin alterations (SCSA—sperm chromatin structure assay) requires a flow cytometer, making it expensive and challenging to apply in field evaluations and assisted reproduction laboratories. This study tested alternative methods based on their efficiency, complexity, and cost, providing veterinarians with a basis for selecting the most appropriate method in each situation. The methods were as follows: difference in staining intensity with toluidine blue (TB) and the Feulgen reaction (FR), assessed both visually and computationally; visual evaluation of smears stained with acridine orange (AO) using epifluorescence and confocal microscopy; and evaluation using transmission electron microscopy (TEM). Among all the methods tested, AO staining and TEM were the most effective for identifying changes in chromatin integrity that interfere with in vitro production efficiency. The results of this study may reinforce the importance of employing techniques to identify chromatin alterations in bull sperm chromatin for selecting fertile animals, as sperm chromatin alterations in bulls are underexplored in breeding programs.

Sperm chromatin analysis is a crucial tool for investigating fertility in bulls, both in the field and in in vitro fertilization. This study aimed to identify efficient, low-cost, and easy-to-apply methods for detecting chromatin integrity alterations. Frozen semen samples from four bulls with varying results in in vitro embryo production (IVEP) were used. The sperm chromatin from these samples was evaluated using different methods: differences in the staining intensity with toluidine blue and the Feulgen reaction (FR), assessed both visually and computationally; visual evaluation of smears stained with acridine orange (AO) using epifluorescence and confocal microscopy; and evaluation using transmission electron microscopy (TEM) of sections positively stained with uranyl and lead. The same samples were utilized for IVEP. The results obtained using each method were correlated with one another and with the cleavage and embryonic development rates achieved in IVEPs. The odds ratio was employed to compare the chromatin alteration identification rates of the studied methods. Overall, chromatin integrity alterations identified using the AO and TEM methods exhibited correlations with the cleavage (r = −1.00) and embryonic development rates (r = −1.00), respectively. Among the methods tested, TEM and AO were the most effective for identifying chromatin integrity alterations that interfere with IVEP efficiency.