Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility

Boar semen from a heterospermic mating trial and semen cryopreserved by various methods were evaluated by the flow cytometric sperm chromatin structure assay (SCSA), which measures the susceptibility of sperm nuclear DNA to acid-induced denaturation in situ. Spermatozoa were treated with a pH 1.4 buffer and then stained with the metachromatic dye acridine orange. Acridine orange intercalated into double-stranded DNA (native) fluoresces green while single-stranded DNA (denatured) fluoresces red when excited with 488 nm light. The ratio of red to total fluorescence provides an index of normality/abnormality. The SCSA data on neat boar semen or semen in either Kiev-Merck or Pursel-Johnson extender and frozen directly on dry ice blocks or plunged into LN(2) did not differ within individual boars. Therefore, chromatin structure, as measured by the SCSA, was not influenced differently by these 2 methods of semen cryopreservation. When semen from 6 boars was mixed in equal sperm numbers in six 3-way combinations and inseminated into at least 3 Duroc gilts per combination, 4 of the 6 combinations yielded 2 litters, while the remaining 2 combinations yielded 3 litters. The SCSA correctly predicted both the high and low fertility boars based on a ratio of offspring as deviated from the theoretical percentage. Thus, the SCSA was found to be a valuable adjunct method for evaluating boar cemen quality.