Sister chromatid exchange induction in sheep peripheral blood mononuclear cells by helio-neon laser radiation.

The effects of laser light on the cellular DNA have not been extensively characterized. Low-power laser sources, such as the helium-neon (He-Ne) laser with a wavelength of 632.8 nm, have been found to produce photobiological and photodamage effects with evidence of interference with cell replication. We have investigated the effects of He-Ne laser irradiation on sister chromatid exchange (SCE) frequencies in sheep peripheral blood mononuclear cells (PBMC). Cultured cells were irradiated once at 6 selected energy intensities of laser irradiation and then stimulated with pokeweed mitogen and cultured in the presence of 5-bromodeoxyuridine (BrdUrd). The frequency of SCEs of both irradiated and non-irradiated cells were analyzed. The mean SCE of irradiated cells significantly increased with growing energy density up to a laser dose of 24 J/cm2, whereas after an energy density of 24 J/cm2, the SCE frequency decreased with increasing energy densities. We concluded that the application of He-Ne laser irradiation at energy densities ranging from 2 to 96 J/cm2 produced a different effect on SCE frequency in sheep PBMC in vitro.

Biological effects of helium-neon (He-Ne) laser irradiation on acrosome reaction in bull sperm cells.

The purpose of this study was to determine the effect of He-Ne laser irradiation (632.8 nm, 10 mW) on the induction of acrosome reaction and mortality in bull sperm cells in comparison with two important capacitation agents; calcium and heparin. Frozen-thawed bull sperm cells were washed in percoll gradient and suspended at a concentration of 1 x 10(6) ml-1 in sp-TALP medium, capacitated in the presence of 2 mM CaCl2, 10 micrograms ml-1 heparin, or irradiated at fluences from 2 to 16 J cm-2, and incubated for 0, 30, 60 and 90 minutes. At the end of the incubation period, the percentage of sperm that were acrosome-reacted and dead was determined. The results obtained indicated that laser irradiation at all fluences produced a significant increase (p < 0.001) in the percentage of sperm cells that were acrosome reacted, and a significant decrease (p < 0.001) in the percentage of dead sperm at 90 minutes of incubation in comparison to other capacitation agents and the control group. The percentage of sperm cells with acrosome reaction was increased with increasing fluences of laser irradiation and time of incubation. It is conclude that the application of He-Ne laser irradiation at fluences from 2 to 16 J cm-2 induced the acrosome reaction and decreased the sperm mortality percentage in vitro of bull sperm cells.

Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro.

The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39 degrees C in 5% CO2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37 degrees C or 39 degrees C) in 5% CO2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37 degrees C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes.

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro.

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.

Cytogenetic study of in vitro-derived bovine embryos.

A cytogenetic study of early bovine embryos (2-16 blastomeres) produced in vitro was conducted to determine the incidence of embryos carrying chromosome anomalies. The embryos were produced from immature oocytes matured in vitro and fertilized by sperm prepared using the Percoll density gradient method. Slides were prepared according to an 'air drying' technique and the chromosomal complement of embryos was studied by Giemsa-staining. Approximately 57% of prepared embryos were suitable for analysis. The results revealed that 18% of cytogenetically analysed embryos presented chromosomal anomalies, including haploidy (8%), aneuploidy (2%) and polyploidy (8%). Our results were compared to the results of other studies in cattle and other domestic animals.

Helium-neon (he-ne) laser irradiation increases the incidence of unreduced bovine oocytes during the first meiotic division in vitro.

Low power He-Ne laser irradiation was applied to immature bovine oocytes to investigate the incidence of unreduced oocytes (diploid oocytes) during the first meiotic division in vitro. Immature bovine oocytes from cows killed at a slaughterhouse were irradiated with He-Ne laser irradiation at 0.05 and 0.25 J/cm(2) exposures. An oocyte group was left untreated serving as the control group. All oocytes were matured in TCM-199 medium supplemented with 10% fetal calf serum (FCS). Unreduced oocyte percentages obtained in the irradiated oocytes group were significantly higher (p<0.01 and p<0.001, respectively) than those of the control group. Furthermore, the laser-treated oocytes showed a degeneration rate significantly higher (p<0.01 and p<0.001, respectively) than those observed in the control group. It is concluded that the application of He-Ne laser irradiation at 0.05 and 0.25 J/cm(2) exposures increased the incidence of unreduced oocytes and the percentage of degenerated oocytes during the in vitro meiosis process of immature bovine oocytes.

The effect of bovine amniotic fluid on in vitro maturation of bovine oocytes.

Three experiments were carried out to investigate whether bovine amniotic fluid (BAF), when added as a protein supplement to the maturation medium, supports the in vitro maturation of pre-ovulatory bovine oocytes. In the first experiment, the medium was supplemented with three different concentrations of the BAF (10, 20 and 50%, respectively). In the second experiment, the effects of a combination of two protein supplements BAF plus oestrus cow serum (BAF-OCS) and BAF plus bovine follicular fluid (BAF-BFF), on the in vitro oocyte maturation rates were evaluated. In the third experiment, the effects of three different protein supplements (BAF, OCS, and BFF) on the oocyte maturation percentages were compared. Maturation rates at the metaphase II stage, following supplementation with 20% BAF (77%) and 10% BAF (68%) were significantly higher (P < 0.001 and P < 0.05, respectively) than those resulting from supplementation with 50% BAF (44%). Significant differences between the 10% BAF and 20% BAF treatments were not observed. Significant differences were, however, found between the BAF-OCS treatment (57%) and the BAF-BFF treatment (50%). The results obtained from the BAF and the OCS treatments (77 and 78%, respectively) were significantly higher (P < 0.05) than when BFF (60%) was used. It is concluded that supplementation of the in vitro maturation medium of pre-ovulatory bovine oocytes with BAF provides important levels of proteins and growth factors responsible for nuclear and cytoplasmic development.