Investigating nuclear shape, morphology, and molecular changes in semen from scrotal-insulated Holstein bulls using Fourier harmonic analysis, flow cytometry, and in vitro fertility evaluations

The objective of this study was to investigate the use of Fourier Harmonic Analysis (FHA) on determining nuclear shape changes in sperm from bulls that had undergone a heat stress event and whether FHA could detect ejaculates with uncompensable defects. The first aim established a heat stress model in the bovine by employing scrotal insulation of five bulls and then using the ejaculates in an in vitro fertility evaluation to determine rates of fertilization, 32/48 hr early cleavage, nuclei numbers at 135 hours post-fertilization, and sperm morphology. Ejaculates were collected 3X a week from each bull, over the entire 65-day period of spermatogenesis, and included a three-week time period prior, to the 48-hour scrotal insulation insult, as a control. The second aim investigated whether changes in sperm nuclear shape, as determined by FHA, corresponded to changes in in vitro fertility and to sperm morphology. The third aim investigated the relationship of sperm nuclear shape change to the incidence of persistent DNA damage, from abortive apoptosis, and two other apoptotic markers occurring in the testes during scrotal insulation and to compare this to changes in in vitro fertility and to sperm morphology. We examined 50,839 embryos and presumptive zygotes and found decreases (p < 0.05) in fertilization, early cleavage, and nuclear numbers reflecting damage in spermatogenesis from late meiosis and continuing through midspermiogenesis. Abnormal sperm head morphology also increased (p < 0.05) during this same period with pyriform, vacuoles, and detached predominating. Fourier Harmonics also detected nuclear shape changes (p < 0.05) beginning in late meiosis and into midspermiogenesis. The HAO and HA2 were the strongest predictors (p < 0.05) of changes in the in vitro fertility evaluation. DNA damage was associated with decreases in in vitro fertility (p < 0.05) and to changes in nuclear shape (p < 0.05) but did not explain all nuclear shape changes. Mitochondria] viability and membrane integrity were decreased (p < 0.05) on days 19 and 33 and corresponded to increases in DNA damage and changes in nuclear shape (p < 0.05). Fourier Harmonics were able to predict changes in in vitro fertility related to changes in sperm nuclear shape.