Technique comparison to assess sperm DNA fragmentation in hair ram

Resumen

Objective: To evaluate 5 DNA fragmentation techniques in thawed sperm samples from hair ram subjected to fragmentation with H2O2.
Design/methodology/approach: Samples were 36 straws from 4 Blackbelly rams that were thawed, mixed (pool), diluted in PBS to a concentration of  30X10⁶ sperm/mL and divided into three treatments: T0: sample without oxidant (considered as 0% damaged DNA), T100: sample incubated with 300 M H2O2 for 24 hours (induction of DNA fragmentation (100%)). Subsequently, half of T0 and T100 were mixed to obtain a proportion of 50% sperm with fragmented DNA (T50). The samples were analyzed with different techniques: Aniline Blue (AB), Toluidine Blue (TB), Acridine Orange (AO), Chromomycin A3 (CMA3) and Sperm Chromatin Dispersion (SCD).
Results: In the linear regression, all the techniques presented a significance level of less than 5%, as well as a significant correlation (r=0.962, P<0.01). However, between treatments, it was observed that the AO technique (34.82 31 ± 3.00%) at T50 and the AB technique (9.55 ± 1.45% at T100 were the least sensitive in detecting DNA damage compared to the other techniques.
Limitations on study/implications: New techniques are increasingly. Findings/conclusions: The techniques that best evaluate the DNAFI of sperm in hair ram are CMA3, SCD and TB.