The effect of low-level laser irradiation on sperm motility, and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm

Abstract

Background and objective: Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm.

Study design/materials and methods: We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS--Integrated Semen Analysis System.

Results: Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05).

Conclusion: We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was more effective than a dose of 6 joules in preserving sperm motility.

Fig 1. Representative Low Level Laser Irradiation in joules 4 and 6 and analyzed by flow cytometry.

(A) Shows the percentage of sperm with acrosome integrity under the total of living cells. (Newman-Keuls Test * p < 0.05). (B) Shows the percentage of live sperm cells relative to the total cells analyzed. (C) Shows representative plots of the respective conditions when exposed to 7AAD and FITC-PSA. The quadrants indicate: UL—sperm dead (plasmatic membrane injured (MP) and acrosome membrane integrate (MA), RH—live sperm (plasmatic membrane injured (MP) and acrosome membrane integrate); LL—live (plasmatic membrane integrate (MP) and Membrane damaged acrosome (MA); LR—(plasmatic membrane injured (MP) and acrosome membrane integrate (MA), in the control, 4 joule and 6 joules groups.

Fig 2. Evaluation of sperm movement through the Integrated Semen Analysis System in (A) Straightness Index percentage of sperm analysis presenting the non-irradiated control group and groups subjected to irradiation with low-power laser with dose 4:06 joules.

Data shown are the mean ± standard deviation. One-way ANOVA and Newman-Keuls post hoc analysis were applied. * p < 0.05 control group vs. 4 Joules group and # p < 0.05. group 6 Joules vs. 4 Joules group. (B) Mobile percentage of progressive sperm analysis presenting the group not irradiated groups and subjected to low-level laser irradiation dose 4 and 6 joules group. Data shown are the mean ± standard deviation. One-way ANOVA and Newman-Keuls post hoc analysis were applied. * p <0.05 control group vs. 4 Joules group. (C) Graphical representation of sperm movement by Integrated Semen Analysis System, The red lines show the movements of the sperm in nm/s and the yellow lines represent the static sperm.