Hoechst 33342 stain and u.v. laser exposure do not induce genotoxic effects in flow-sorted boar spermatozoa

Abstract

Sex selection by flow cytometry/cell sorting involves the staining of spermatozoa with Hoechst 33342 in combination with the impact of a u.v. laser beam, two potentially mutagenic agents. A phenotypic and cytogenetic study of lymphocytes of piglets born after insemination with spermatozoa stained with Hoechst 33342 and from piglets obtained from stain-sorted spermatozoa was performed to evaluate the genotoxic effect of Hoechst 33342 staining and u.v. laser irradiation on the offspring. Lymphocytes from piglets born after insemination with unstained spermatozoa, but from the same ejaculate, were used as a control group. Peripheral blood lymphocytes from these piglets were cultured following a standard cell culture protocol. Cells were then collected by centrifugation, subjected to hypotonic solution and fixed and dropped onto slides. Sister chromatid exchanges (SCEs) and chromosome aberrations (CAs: including chromosome and chromatid breaks) per cell were scored in 50-s division metaphase spreads from each donor. Reproductive parameters and litter performance of all inseminations performed were also recorded in all groups. Data were analyzed by ANOVA. No significant increase (P > 0.05) of SCE and CA frequencies were observed in piglets born from stained spermatozoa or from stain-sorted spermatozoa with respect to controls (untreated sperm). The results indicated that no mutagenic effect on spermatozoa, expressed as increases in the incidence of abnormalities in the resulting offspring and also as increases in SCE and CA frequencies on lymphocytes from these individuals, was induced by the staining of boar spermatozoa with Hoechst 33342, nor by combination of staining with laser impact during flow cytometry.

Sex Preselection in Bovine by Separation of X- and Y-Chromosome Bearing Spermatozoa

“…After Hoechst-stained and UV-laser irradiated sperm nuclei were microinjected into hamster eggs, the n u m be r o f D N A b re a k s a n d t r i r a d i a l a n d quadriradial exchanges between the chromosomes that developed increased slightly. In a recent study, no genotoxic effects of sorting sperm with Hoechst 33342 were found in the cells of resulting pigs [41].…”

Partial protection of N-acetylcysteine against MPA-capped CdTe quantum dot-induced neurotoxicity in rat primary cultured hippocampal neurons

“…Hoechst 33342, a type of blue- fluorescent dye, stains the condensed chromatin in apoptotic cells more brightly than normal chromatin, 38 while propidium iodide (PI), a red-fluorescent dye, only permeates into dead cells. Hoechst 33342, a type of blue- fluorescent dye, stains the condensed chromatin in apoptotic cells more brightly than normal chromatin, 38 while propidium iodide (PI), a red-fluorescent dye, only permeates into dead cells.…”

Birth of puppies of predetermined sex after artificial insemination with a low number of sex‐sorted, frozen–thawed spermatozoa in field conditions

“…Damage due to sexing sperm has also been cited as a reason for lower pregnancy rates (Seidel ; Parrilla et al . ). Despite the damage to the sexed sperm, which contributes to the lower fertility, the resulting offspring appear to be completely normal (Tubman et al .…”

“…No genotoxic effects of sexing the sperm with Hoechst 33342 have been found in cells in prior studies (Morrell & Dresser ; Parrilla et al . ), but staining with Hoechst 33342 and UV laser irradiation of the sperm during flow sorting tended to increase the incidence of chromosomal aberrations (Garner ). Damage due to sexing sperm has also been cited as a reason for lower pregnancy rates (Seidel ; Parrilla et al .…”