309 DIFFERENCES IN PRONUCLEUS FORMATION RATES BETWEEN BULLS IN RESPONSE TO GAMMA IRRADIATION OF FROZEN–THAWED SEMEN

Abstract

Gamma irradiation induces DNA damage to mature bovine spermatozoa but does not affect motility, membrane integrity, or function (Fatehi et al. 2006 J. Androl. 27, 176–188), making it a useful model to evaluate the effect of fertilization with DNA-damaged spermatozoa. The objective of the present study was to analyze the fertilizing capacity of frozen–thawed gamma-irradiated sperm from 2 bulls: a bull with a high sperm quality and fertilization rate (bull A), and a bull with a low sperm quality and fertilization rate (bull B). To ensure that DNA damage was induced, frozen–thawed semen was exposed to a high dose of irradiation (90 Gy). Cumulus–oocyte complexes were obtained from abattoir ovaries and were in vitro-matured (IVM) using standard procedures (23 h in DMEM with 5% serum and eCG/hCG). For each treatment group, 3 to 5 straws of semen from the same ejaculate were used. After thawing, semen from the 2 bulls was either irradiated or held at room temperature before 1:1 dilution in sperm-TALP. During irradiation, semen was kept in the straws. The irradiated and nonirradiated sperm were used for IVF (23 h in IVF-TALP) within 1 h of thawing. IVM and IVF were carried out at 38.5°C in 5% CO₂ in air. After IVF, presumptive zygotes were whole mount-fixed and 24 h later were stained with 1% aceto-orcein followed by determination of fertilization status. Fertilization was considered normal if 2 pronuclei (PN) were present. A total of 4 replicates were performed and 286 zygotes analyzed. No difference was detected between replicates, and the results were pooled. Fisher's exact test was used to determine effect of treatment. More zygotes had 2 PN after fertilization with semen from bull A (47/67 = 70%), compared with that from bull B (16/71 = 23%; P < 0.0001) using nonirradiated sperm. Irradiation of sperm significantly increased the fraction of zygotes with 2 PN from bull A (61/70 = 87%; P = 0.03), but decreased the 2 PN fractions in zygotes fertilized with sperm from bull B (2/75 = 3%; P = 0.0002). Ideally, to avoid straw variation, semen should have been pooled and divided into groups before irradiation. The variation between straws might explain the higher fertilization rates in bull A using irradiated sperm compared with nonirradiated sperm. In conclusion, there appear to be differences in fertilizing ability between bulls after irradiation of frozen–thawed sperm. This could be due to suboptimal DNA packaging, which made sperm from bull B more susceptible to radiation-induced damage. The potential irradiation-induced increase in DNA fragmentation in sperm from bull B compared with that from bull A might delay or prevent the formation of the 2 PN. Further studies are needed to investigate differences in fertilization and early embryonic development using sperm with intact or damaged DNA.