Sperm chromatin stability in frozen-thawed semen is maintained over age in AI bulls

The aim of the present study was to investigate the effect of age of the sire on the in vitro quality of frozen-thawed (FT) bull spermatozoa, both when tested immediately postthaw (PT) and when assessed after cleansing and selection through a swim-up (SU) procedure. Semen samples from six Swedish Red and White Breed (SRB) artificial insemination (AI) bulls at age 1 and again, at 4 years were collected and frozen in 0.25 ml plastic straws. Also, semen was collected from six Estonian Holstein (EHF) bulls at the ages of 3, 5, and 7 years and likewise processed. The FT semen was tested for the susceptibility of sperm nuclear deoxyribonucleic acid (DNA) to undergo acid-induced denaturation in situ, as quantified by flow cytometry (FCM). The DNA denaturability was expressed as function alpha t, i.e., as the ratio of red (denaturated DNA) to red + green (total cellular DNA) fluorescence intensity. The results were expressed as the percentage of cells with high alpha t values, i.e., cells outside the main population (% COMP alpha t). Morphological evaluation of the same samples was performed to detect general and sperm head abnormalities and differences between ages. Fertility results were available as non-return rates (NRRs) for the semen of the sires when they were 1 year (SRB) and 3 years (EHF) old, varying from 62.2 to 70.7% in SRB and from 52.2 to 76.0% in EHF animals. The COMP alpha t values ranged from 0.5-3.6% (PT) to 0.2-1.7% (SU) for SRB bulls and from 0.4-1.8% (PT) to 0.2-1.5% (SU) for EHF bulls. Both breeds lacked differences between ages, either PT or after SU. However, the SU procedure yielded a significantly higher population of spermatozoa with stable DNA following acid-induced denaturation, than PT samples (p < 0.001). No correlation was detected between field fertility and chromatin stability. The results indicate that for these bull populations, the SU procedure was able to select spermatozoa with stable chromatin from the bulk samples. However, the use of DNA denaturation as a challenge to assess sperm chromatin stability did not offer a more accurate tool to evaluate sperm quality than the conventional, light microscopical evaluation of morphology.